Stat3 pathway inhibitors and cancer stem cell inhibitors

ABSTRACT

The present invention relates to a novel naphtho class of compounds as Stat3 pathway inhibitors and as cancer stem cell inhibitors; to methods of using such compounds to treat cancer; to methods of using such compounds to treat disorders in a mammal related to aberrent Stat3 pathway activity; to pharmaceutical compositions containing such compounds.

CLAIM OF PRIORITY

This application is a continuation of U.S. patent application Ser. No.15/429,939, filed on Feb. 10, 2017, which is a continuation of U.S.patent application Ser. No. 14/499,299, filed on Sep. 29, 2014, which isa continuation of U.S. patent application Ser. No. 12/677,511, filed onJan. 3, 2011, which is a U.S. national stage application filed under 35U.S.C. § 371 from International Application Serial No.PCT/US2008/075848, filed on Sep. 10, 2008, which claims the benefit ofpriority to U.S. Provisional Application Ser. No. 61/013,372, filed onDec. 13, 2007 and U.S. Provisional Application Ser. No. 60/971,144,filed on Sep. 10, 2007. The contents of the above applications areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to a novel class of compounds as Stat3pathway inhibitors, cancer stem cell inhibitors as well as cancer stemcell pathway inhibitors; to methods of using such compounds to treatcancer; to methods of using such compounds to treat disorders in amammal related to aberrent Stat3 pathway activity; to synthesis andpharmaceutical compositions containing such compounds.

BACKGROUND OF THE INVENTION

Introduction of Stat3 Pathway. Stat3 is a member of the Stat familywhich are latent transcription factors activated in response tocytokines/growth factors to promote proliferation, survival, and otherbiological processes. Stat3 is activated by phosphorylation of acritical tyrosine residue mediated by growth factor receptor tyrosinekinases, Janus kinases, and/or the Src family kinases, etc. Thesekinases include but not limited to EGFR, JAKs, Abl, KDR, c-Met, Src, andHer2 [1]. Upon tyrosine phosphorylation, Stat3 forms homo-dimers andtranslocates to the nucleus, binds to specific DNA-response elements inthe promoters of the target genes, and induces gene expression [2].

Importance of Stat3 pathway in Targeting Conventional Aspects ofCancers. In normal cells, Stat3 activation is transient and tightlyregulated, lasting from 30 minutes to several hours. However, Stat3 isfound to be aberrantly active in a wide variety of human cancers,including all the major carcinomas as well as some hematologic tumors.Stat3 plays multiple roles in cancer progression. As a potenttranscription regulator, it targets genes involved in cell cycle, cellsurvival, oncogenesis, tumor invasion, and metastasis, such as Bcl-x1,c-Myc, cyclin D1, Vegf, MMP-2, and survivin [3-8]. It is also a keynegative regulator of tumor immune surveillance and immune cellrecruitment [9-11].

Ablating Stat3 signaling by antisense, siRNA, dominant-negative form ofStat3, and/or blockade of tyrosine kinases causes cancer cell-growtharrest, apotosis, and reduction of metastasis frequency in vitro and/orin vivo [2, 4, 12, 13].

Importance of Stat3 pathway in Other Diseases. Activation of Stat3 byvarious cytokines, such as Interleukin 6 (IL6) has been demonstrated ina number of autoimmune and inflammatory diseases. Recently, it has beenrevealed that the Stat3 pathway promotes pathologic immune responsesthrough its essential role in generating TH17 T cell responses [14]. Inaddition, Stat3 pathway mediated inflammation is the common causativeorigin for atherosclerosis, peripheral vascular disease, coronary arterydisease, hypertension, osteroprorosis, type 2 diabetes, and dementia.Therefore, Stat3 inhibitors may be used to prevent and treat autoimmuneand inflammatory diseases as well as the other diseases listed abovethat are caused by inflammation.

Introduction of Cancer Stem cells (CSCs). Cancer stem cells (CSCs) are asub-population of cancer cells (found within tumors or hematologicalcancers) that possess characteristics normally associated with stemcells. These cells are tumorigenic (tumor-forming), in contrast to thebulk of cancer cells, which are non-tumorigenic. In human acute myeloidleukemia the frequency of these cells is less than 1 in 10,000 [15].There is mounting evidence that such cells exist in almost all tumortypes. However, as cancer cell lines are selected from a sub-populationof cancer cells that are specifically adapted to growth in tissueculture, the biological and functional properties of these cell linescan change dramatically. Therefore, not all cancer cell lines containcancer stem cells.

CSCs have stem cell properties such as self-renewal and the ability todifferentiate into multiple cell types. They persist in tumors as adistinct population and they give rise to the differentiated cells thatform the bulk of the tumor mass and phenotypically characterize thedisease. CSCs have been demonstrated to be fundamentally responsible forcarcinogenesis, cancer metastasis, and cancer reoccurrence. CSCs arealso often called tumor initiating cells, cancer stem-like cells,stem-like cancer cells, highly tumorigenic cells, or super malignantcells.

Clinical Implications of Cancer Stem Cells. The existence of cancer stemcells has several implications in terms of cancer treatment and therapy.These include disease identification, selective drug targets, preventionof cancer metastasis and recurrence, treatment of cancer refractory tochemotherapy and/or radiotherapy, treatment of cancers inherentlyresistant to chemotherapy or radiotherapy and development of newstrategies in fighting cancer.

The efficacy of cancer treatments are, in the initial stages of testing,often measured by the amount of tumor mass they kill off. As CSCs wouldform a very small proportion of the tumor and have markedly differentbiologic characteristics than their differentiated progeny, themeasurement of tumor mass may not necessarily select for drugs that actspecifically on the stem cells. In fact, cancer stem cells areradio-resistant and also refractory to chemotherapeutic and targeteddrugs. Normal somatic stem cells are naturally resistant tochemotherapeutic agents—they have various pumps (such as MDR) thatefflux drugs, higher DNA repair capability, and have a slow rate of cellturnover (chemotherapeutic agents naturally target rapidly replicatingcells). Cancer stem cells, being the mutated counterparts of normal stemcells, may also have similar functions which allow them to survivetherapy. In other words, conventional chemotherapies kill differentiatedor differentiating cells, which form the bulk of the tumor that areunable to generate new cells. A population of cancer stem cells whichgave rise to it could remain untouched and cause a relapse of thedisease. Furthermore, treatment with chemotherapeutic agents may onlyleave chemotherapy-resistant cancer stem cells, so that the ensuingtumor will most likely also be resistant to chemotherapy. Cancer stemcells have also been demonstrated to be resistant to radiotherapy (XRT)[16, 17].

Since surviving cancer stem cells can repopulate the tumor and causerelapse, it would be possible to treat patients with aggressive,non-resectable tumors and refractory or recurrent cancers, as well asprevent the tumor metastasis and recurrence by selectively targetingcancer stem cells. Development of specific therapies targeted at cancerstem cells therefore holds hope for improvement of survival and qualityof life of cancer patients, especially for sufferers of metastaticdisease. The key to unlocking this untapped potential is theidentification and validation of pathways that are selectively importantfor cancer stem cell self-renewal and survival. Though multiple pathwaysunderlying tumorigenesis in cancer and in embryonic stem cells or adultstem cells have been elucidated in the past, no pathways have beenreported for cancer stem cell self-renewal and survival.

Identification and Isolation of CSCs. The methods on identification andisolation of cancer stem cells have been reported. The methods are usedmainly to exploit the ability of CSCs to efflux drugs, or are based onthe expression of surface markers associated with cancer stem cells.

SCs are resistant to many chemotherapeutic agents, therefore it is notsurprising that CSCs almost ubiquitously overexpress drug efflux pumpssuch as ABCG2 (BCRP-1) [18-22], and other ATP binding cassette (ABC)superfamily members [23, 24]. The side population (SP) technique,originally used to enrich hematopoetic and leukemic stem cells, wasfirst employed to identify CSCs in the C6 glioma cell line [25]. Thismethod, first described by Goodell et al., takes advantage ofdifferential ABC transporter-dependent efflux of the fluorescent dyeHoechst 33342 to define a cell population enriched in CSCs [21, 26]. TheSP is revealed by blocking drug efflux with verapamil, so that the SP islost upon verapamil addition.

Efforts have also focused on finding specific markers that distinguishcancer stem cells from the bulk of the tumor. Markers originallyassociated with normal adult stem cells have been found to also markcancer stem cells and co-segregate with the enhanced tumorigenicity ofCSCs. The most commonly expressed surface markers by the cancer stemcells include CD44, CD133, and CD166 [27-33]. Sorting tumor cells basedprimarily upon the differential expression of these surface marker(s)have accounted for the majority of the highly tumorigenic CSCs describedto date. Therefore, these surface markers are well validated foridentification and isolation of cancer stem cells from the cancer celllines and from the bulk of tumor tissues.

SUMMARY

We have identified Stat3 as a key cancer stem cell survival andself-renewal pathway. Therefore, Stat3 pathway inhibitors can killcancer stem cells and inhibit cancer stem cell self-renewal.

In one aspect, the present invention provides a compound of formula I,

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptablesalt or solvate thereof, wherein the symbols have the following meaningsand are, for each occurrence, independently selected:

-   X is O or S;-   R₁ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkynyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a);-   R₃ is hydrogen, cyano, CF₃, OCF₃, alkyl or substituted alkyl,    alkenyl or substituted alkenyl, alkynyl or substituted alkynyl,    cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted    cycloalkenyl, heterocycle or substituted heterocycle, aryl or    substituted aryl, OR_(a), SR_(a), or NR_(b)R_(c);-   R₇ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkynyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a);-   R_(a) is hydrogen, alkyl or substituted alkyl, alkenyl or    substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or    substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl,    heterocycle or substituted heterocycle, or aryl or substituted aryl;-   R_(b) and R_(c) are independently hydrogen, alkyl or substituted    alkyl, cycloalkyl or substituted cycloalkyl, heterocycle or    substituted heterocycle, or aryl or substituted aryl, or said R_(b)    and R_(c) together with the N to which they are bonded optionally    form a heterocycle or substituted heterocycle; and-   n is 1-4,-   provided that when R₃ is not NR_(b)R_(c), then R₇ is not hydrogen    and at least one of R₁ and R₇ is halogen, aryl, or substituted aryl.

In another aspect, the present invention provides a compound of formulaII,

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptablesalt or solvate thereof, wherein the symbols have the following meaningsand are, for each occurrence, independently selected:

-   X is O or S;-   R₁ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkynyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, or SR_(a);-   R₄ is hydrogen, alkyl or substituted alkyl, alkenyl or substituted    alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted    cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle or    substituted heterocycle, aryl or substituted aryl, alkylaryl or    substituted alkylaryl;-   R₅ is hydrogen, alkyl or substituted alkyl, cycloalkyl or    substituted cycloalkyl, aryl or substituted aryl, alkylaryl or    substituted alkylaryl; optionally, R₄ and R₅ may be combined to form    alkenyl or substituted alkenyl;-   R₇ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkynyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a);

R_(a) is hydrogen, alkyl or substituted alkyl, alkenyl or substitutedalkenyl, alkynyl or substituted alkynyl, cycloalkyl or substitutedcycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle orsubstituted heterocycle, or aryl or substituted aryl; and

-   n is 1-4,-   provided that at least one of R₁ and R₇ is halogen; or at least one    of R₁, R₄, R₅ and R₇ is aryl or substituted aryl.

In yet another aspect, the present invention provides a compound offormula III,

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptablesalt or solvate thereof, wherein the symbols have the following meaningsand are, for each occurrence, independently selected:

-   X is O or S;-   R₁ is halogen;-   R₄ is hydrogen, alkyl or substituted alkyl, alkenyl or substituted    alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted    cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle or    substituted heterocycle, aryl or substituted aryl, alkylaryl or    substituted alkylaryl;-   R₅ is hydrogen, alkyl or substituted alkyl, cycloalkyl or    substituted cycloalkyl, aryl or substituted aryl, alkylaryl or    substituted alkylaryl; optionally, R₄ and R₅ may be combined to form    alkenyl or substituted alkenyl;-   R₆ is hydrogen, alkyl or substituted alkyl, OR_(a), OC(═O)R_(a), or    SR_(a);-   R₇ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkynyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a);-   R_(a) is hydrogen, alkyl or substituted alkyl, alkenyl or    substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or    substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl,    heterocycle or substituted heterocycle, or aryl or substituted aryl;    and-   n is 1-4.

In yet another aspect, the present invention provides a compound offormula IV,

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptablesalt or solvate thereof, wherein the symbols have the following meaningsand are, for each occurrence, independently selected:

-   X is O or S;-   R₁ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkynyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a);-   R₇ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkynyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a);-   R₈ is hydrogen, alkyl or substituted alkyl, cycloalkyl or    substituted cycloalkyl, aryl or substituted aryl, alkylaryl or    substituted alkylaryl;-   R₉ and R¹⁰ are each independently hydrogen, alkyl or substituted    alkyl, cycloalkyl or substituted cycloalkyl, aryl or substituted    aryl, heterocycle or substituted heterocycle, alkylaryl or    substituted alkylaryl, alkylheteroaryl or substituted    alkylheteroaryl; or R₉ and R₁₀ together with the carbon to which    they are bonded optionally form cycloalkyl or substituted    cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle or    substituted heterocycle;-   R_(a), is hydrogen, alkyl or substituted alkyl, alkenyl or    substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or    substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl,    heterocycle or substituted heterocycle, or aryl or substituted aryl;    and-   n is 1-4.

In yet another aspect, the present invention provides a compound offormula V,

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptablesalt or solvate thereof, wherein the symbols have the following meaningsand are, for each occurrence, independently selected:

-   X is O or S;-   R₁ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkynyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a);-   R₇ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkynyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a);-   R₁₁ is hydrogen, alkyl or substituted alkyl, alkenyl or substituted    alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted    cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle or    substituted heterocycle, aryl or substituted aryl, alkylaryl or    substituted alkylaryl, alkylheteroaryl or substituted    alkylheteroaryl;-   R_(a) is hydrogen, alkyl or substituted alkyl, alkenyl or    substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or    substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl,    heterocycle or substituted heterocycle, or aryl or substituted aryl;    and-   n is 1-4.

In yet another aspect, the present invention provides a compound offormula VI,

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptablesalt or solvate thereof, wherein the symbols have the following meaningsand are, for each occurrence, independently selected:

-   X is O or S;-   each R₁ is independently hydrogen, halogen, cyano, nitro, CF₃, OCF₃,    alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkynyl    or substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a);-   R₃ is hydrogen, cyano, CF₃, OCF₃, alkyl or substituted alkyl,    alkenyl or substituted alkenyl, alkynyl or substituted alkynyl,    cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted    cycloalkenyl, heterocycle or substituted heterocycle, aryl or    substituted aryl, OR_(a), SR_(a), or NR_(b)R_(c);-   R₇ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkynyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a);-   R_(a) is hydrogen, alkyl or substituted alkyl, alkenyl or    substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or    substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl,    heterocycle or substituted heterocycle, or aryl or substituted aryl;-   R_(b) and R_(c), are independently hydrogen, alkyl or substituted    alkyl, cycloalkyl or substituted cycloalkyl, heterocycle or    substituted heterocycle, or aryl or substituted aryl, or said R_(b)    and R_(c) together with the N to which they are bonded optionally    form a heterocycle or substituted heterocycle; and provided that    when R₃ is hydroxyl, alkyl, or substituted alkyl, then R₁ is    halogen, aryl, or substituted aryl; and further provided that when    R₃ is aryl or substituted aryl, then R₇ is not hydrogen, and further    provided that 2-acetyl-7-chloro-naphtho[2,3-b]furan-4,9-diene and    2-acetyl-7-fluoro-naphtho[2,3-b]furan-4,9-dione are excluded.

In yet another aspect, the present invention provides a compound offormula VII:

-   R₁ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkynyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a);-   R₃ is hydrogen, cyano, CF₃, OCF₃, alkyl or substituted alkyl,    alkenyl or substituted alkenyl, alkynyl or substituted alkynyl,    cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted    cycloalkenyl, heterocycle or substituted heterocycle, aryl or    substituted aryl, OR_(a), SR_(a), or NR_(b)R_(c);-   R₇ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkynyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a);

R_(a) is hydrogen, alkyl or substituted alkyl, alkenyl or substitutedalkenyl, alkynyl or substituted alkynyl, cycloalkyl or substitutedcycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle orsubstituted heterocycle, or aryl or substituted aryl;

R_(b) and R_(c) are independently hydrogen, alkyl or substituted alkyl,cycloalkyl or substituted cycloalkyl, heterocycle or substitutedheterocycle, or aryl or substituted aryl, or said R_(b) and R_(c)together with the N to which they are bonded optionally form aheterocycle or substituted heterocycle; and

-   n is 1-4,-   provided that when R₃ is not NR_(b)R_(c), then R₇ is not hydrogen.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a compound formulae I-VII as describedhereinabove, or an enantiomer, diastereomer, tautomer, or apharmaceutically-acceptable salt thereof, and apharmaceutically-acceptable excipient, carrier, or diluent.

In yet another aspect, the present invention provides a method oftreating cancer in a mammal, comprising administering to the mammal inneed thereof a therapeutically effective amount of a compound offormulae I-VII as described hereinabove, or an enantiomer, diastereomer,tautomer, or pharmaceutically acceptable salt or solvate thereof. In oneembodiment, the said cancer above is selected from breast cancer, headand neck cancer, lung cancer, ovarian cancer, pancreatic cancer,multiple myeloma, colorectal carcinoma, prostate cancer, melanoma,kaposi sarcoma, ewing's sarcoma, liver cancer, gastric cancer,medulloblastoma, brain tumors, leukemia. In another embodiment, the saidcancer above is selected from lung cancer, breast cancer, cervicalcancer, colon cancer, liver cancer, head and neck cancer, pancreaticcancer, gastric cancer, and prostate cancer.

In another aspect, the present invention provides a method of inhibitingor reducing unwanted Stat3 pathway activity with an effective amount ofa compound of formulae I-VII as described hereinabove, or an enantiomer,diastereomer, tautomer, or pharmaceutically acceptable salt or solvatethereof.

In a further aspect, the present invention provides a method of treatinga disorder associated with aberrant Stat3 pathway activity in a mammal,comprising administering to the mammal in need thereof a therapeuticallyeffective amount of a compound of formulae I-VII as describedhereinabove, or an enantiomer, diastereomer, tautomer, orpharmaceutically acceptable salt or solvate thereof. The aberrant Stat3pathway activity can be identified by expression of phosphorylated Stat3or its surrogate upstream or downstream regulators. In one embodiment,the said disorder is a cancer associated with aberrant Stat3 pathwayactivity which include but not limited to Breast cancer, head and neckcancer, lung cancer, ovarian cancer, pancreatic cancer, colorectalcarcinoma, prostate cancer, renal cell carcinoma, melanoma,hepatocellular carcinomas, cervical cancer, sarcomas, brain tumors,gastric cancers, multiple myeloma, leukemia, and lymphomas. In anotherembodiment of the aspect, the said disorder is an autoimmune orinflammatory diseases associated with aberrant Stat3 pathway activity.

In another aspect, the present invention provides use of a compound offormula VIII:

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptablesalt or solvate thereof, wherein the symbols have the following meaningsand are, for each occurrence, independently selected:

-   X is O or S;-   R₁ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkynyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a);-   R₇ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkynyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a);-   R₁₂ is hydrogen, alkyl or substituted alkyl, alkenyl or substituted    alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted    cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle or    substituted heterocycle, aryl or substituted aryl, alkylaryl or    substituted alkylaryl, —C(═O)R₃, or —C(OH)R₄R₅;-   R₃ is hydrogen, cyano, CF₃, OCF₃, alkyl or substituted alkyl,    alkenyl or substituted alkenyl, alkynyl or substituted alkynyl,    cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted    cycloalkenyl, heterocycle or substituted heterocycle, aryl or    substituted aryl, OR_(a), SR_(a), or NR_(b)R_(c);-   R₄ is hydrogen, alkyl or substituted alkyl, alkenyl or substituted    alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted    cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle or    substituted heterocycle, aryl or substituted aryl, alkylaryl or    substituted alkylaryl;-   R₅ is hydrogen, alkyl or substituted alkyl, cycloalkyl or    substituted cycloalkyl, aryl or substituted aryl, alkylaryl or    substituted alkylaryl; optionally, R₄ and R₅ may be combined to form    alkenyl or substituted alkenyl;-   R_(a) is hydrogen, alkyl or substituted alkyl, alkenyl or    substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or    substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl,    heterocycle or substituted heterocycle, or aryl or substituted aryl;-   R_(b) and are independently hydrogen, alkyl or substituted alkyl,    cycloalkyl or substituted cycloalkyl, heterocycle or substituted    heterocycle, or aryl or substituted aryl, or said R_(b) and R_(c),    together with the N to which they are bonded optionally form a    heterocycle or substituted heterocycle; and-   n is 1-4,-   provided that 2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione,    2-acetyl-7-chloro-naphtho[2,3-b]furan-4,9-dione,    2-acetyl-7-fluoro-naphtho[2,3-b]furan-4,9-dione,    2-acetylnaphtho[2,3-b]furan-4,9-dione, and    2-ethyl-naphtho[2,3-b]furan-4,9-dione are excluded.

In a further aspect, the present invention provides a method ofinhibiting cellular Stat3 pathway activity in a cell, comprisingadministering to the cell in need thereof an effective amount of acompound of formulae as described herein such that at least undesiredStat3 pathway activity in the cell is reduced.

In one aspect, the present invention provides a method of treating adisorder associated with aberrant Stat3 pathway activity in a subject,the method comprising administering to the subject a therapeuticallyeffective amount of a compound of formulae as described herein.

In another aspect, the present invention provides a method of treating apatient, the method comprising: selecting a patient by aberrant Stat3pathway activity; and administering to the patient a therapeuticallyeffective amount of a compound of formulae I-VIII as described herein.

In yet another aspect, the present invention provides a method oftreating a patient tested to have cancer expressing aberrant Stat3pathway activity by administering to the patient a therapeuticallyeffective amount of a compound of formulae I-VIII as described herein.

In yet another aspect, the present invention provides a method ofinhibiting a cancer stem cell survival and/or self-renewal, the methodcomprising administering to a cancer stem cell with an effective amountof a compound of formulae I-VIII as described herein.

In yet another aspect, the present invention provides a method oftreating a subject for cancer refractory to a standard regimen oftreatment, the method comprising administering the subject atherapeutically effective amount of a compound of formulae as describedherein.

In yet another aspect, the present invention provides a method oftreating relapsed cancer in a subject, the method comprisingadministering the subject a therapeutically effective amount of acompound of formulae as described herein.

In yet another aspect, the present invention provides a method oftreating or preventing cancer metastasis in a subject, the methodcomprising administering the subject a therapeutically effective amountof a compound of formulae as described herein.

In yet another aspect, the present invention provides a method oftreating a cancer in a subject, the method comprising administering thesubject a therapeutically effective amount of formulae as describedherein.

Other aspects and embodiments of the present invention are set forth orwill be readily apparent from the following detailed description of theinvention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the Stat3 pathway in cancer.

FIG. 2 shows the cancer stem cell specific and conventional cancertherapies.

FIG. 3A shows that Stat3 is constitutively active in Hoechst SidePopulation cells.

FIG. 3B shows that Stat3 is constitutively active in CD133⁺ cells.

FIG. 4A shows the Stat3 knockdown in cancer stem cells.

FIG. 4B shows that Stat3 knockdown in cancer stem cells inducesapoptosis.

FIG. 5 shows that Stat3 knockdown in cancer stem cells inhibits cancerstem cell spherogenesis.

FIG. 6A shows that compound 401 inhibits Stat3 DNA-binding activity innuclear extract.

FIG. 6B shows that compounds 416 and 18 inhibits Stat3 DNA-bindingactivity in nuclear extract.

DETAILED DESCRIPTION

A. Definitions

The following are definitions of terms used in the presentspecification. The initial definition provided for a group or termherein applies to that group or term throughout the presentspecification individually or as part of another group, unless otherwiseindicated.

The terms “alkyl” and “alk” refers to a straight or branched chainalkane (hydrocarbon) radical containing from 1 to 12 carbon atoms,preferably 1 to 6 carbon atoms. Exemplary “alkyl” groups include methyl,ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl pentyl, hexyl,isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl,nonyl, decyl, undecyl, dodecyl, and the like. The term “C₁-C₄ alkyl”refers to a straight or branched chain alkane (hydrocarbon) radicalcontaining from 1 to 4 carbon atoms, such as methyl, ethyl, propyl,isopropyl, n-butyl, t-butyl, and isobutyl. “Substituted alkyl” refers toan alkyl group substituted with one or more substituents, preferably 1to 4 substituents, at any available point of attachment. Exemplarysubstituents include but are not limited to one or more of the followinggroups: hydrogen, halogen (e.g., a single halogen substituent ormultiple halo substitutents forming, in the latter case, groups such asCF₃ or an alkyl group bearing Cl₃), cyano, nitro, CF₃, OCF₃, cycloalkyl,alkenyl, cycloalkenyl, alkynyl, heterocycle, aryl, OR_(a), SR_(a),S(═O)R_(e), P(═O)₂R_(e), P(═O)₂R_(e), S(═O)₂OR_(e), P(═O)₂OR_(e),NR_(b)R_(c), NR_(b)S(═O)₂R_(e), NR_(b)P(═O)₂R_(e), S(═O)₂NR_(b)R_(c),P(═O)₂NR_(b)R_(c), C(═O)OR_(d), C(═O)R₃, C(═O)NR_(b)R_(c), OC(═O)R_(a),OC(═O)NR_(b)R_(e), NR_(b)C(═O)OR_(e), NR_(d)C(═O)NR_(b)R_(c),NR_(d)S(═O)₂NR_(b)R_(c), NR_(d)P(═O)₂NR_(b)R_(c), NR_(b)C(═O)R_(a), orNR_(b)P(═O)₂R_(e), wherein R_(a) is hydrogen, alkyl, cycloalkyl,alkenyl, cycloalkenyl, alkynyl, heterocycle, or aryl; R_(b), R_(c) andR_(d) are independently hydrogen, alkyl, cycloalkyl, heterocycle, aryl,or said R_(b) and R_(c) together with the N to which they are bondedoptionally form a heterocycle or substituted heterocycle; and R_(e) isalkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, or aryl.In the aforementioned exemplary substitutents, groups such as alkyl,cycloalkyl, alkenyl, alkynyl, cycloalkenyl, heterocycle and aryl canthemselves be optionally substituted.

The term “alkenyl” refers to a straight or branched chain hydrocarbonradical containing from 2 to 12 carbon atoms and at least onecarbon-carbon double bond. Exemplary such groups include ethenyl or al“Substituted alkenyl” refers to an alkenyl group substituted with one ormore substituents, preferably 1 to 4 substituents, at any availablepoint of attachment. Exemplary substituents include, but are not limitedto, alkyl or substituted alkyl, as well as those groups recited above asexemplary alkyl substituents. The exemplary substitutents can themselvesbe optionally substituted.

The term “alkynyl” refers to a straight or branched chain hydrocarbonradical containing from 2 to 12 carbon atoms and at least one carbon tocarbon triple bond. Exemplary such groups include ethynyl. “Substitutedalkynyl” refers to an alkynyl group substituted with one or moresubstituents, preferably 1 to 4 substituents, at any available point ofattachment. Exemplary substituents include, but are not limited to,alkyl or substituted alkyl, as well as those groups recited above asexemplary alkyl substituents. The exemplary substitutents can themselvesbe optionally substituted.

The term “cycloalkyl” refers to a fully saturated cyclic hydrocarbongroup containing from 1 to 4 rings and 3 to 8 carbons per ring.Exemplary such groups include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, etc. “Substituted cycloalkyl” refers to acycloalkyl group substituted with one or more substituents, preferably 1to 4 substituents, at any available point of attachment. Exemplarysubstituents include, but are not limited to, nitro, cyano, alkyl orsubstituted alkyl, as well as those groups recited above as exemplaryalkyl substituents. The exemplary substitutents can themselves beoptionally substituted. Exemplary substituents also includespiro-attached or fused cylic substituents, especially spiro-attachedcycloalkyl, spiro-attached cycloalkenyl, spiro-attached heterocycle(excluding heteroaryl), fused cycloalkyl, fused cycloalkenyl, fusedheterocycle, or fused aryl, where the aforementioned cycloalkyl,cycloalkenyl, heterocycle and aryl substitutents can themselves beoptionally substituted.

The term “cycloalkenyl” refers to a partially unsaturated cyclichydrocarbon group containing 1 to 4 rings and 3 to 8 carbons per ring.Exemplary such groups include cyclobutenyl, cyclopentenyl, cyclohexenyl,etc. “Substituted cycloalkenyl” refers to a cycloalkenyl groupsubstituted with one more substituents, preferably 1 to 4 substituents,at any available point of attachment. Exemplary substituents include butare not limited to nitro, cyano, alkyl or substituted alkyl, as well asthose groups recited above as exemplary alkyl substituents. Theexemplary substitutents can themselves be optionally substituted.Exemplary substituents also include spiro-attached or fused cylicsubstituents, especially spiro-attached cycloalkyl, spiro-attachedcycloalkenyl, spiro-attached heterocycle (excluding heteroaryl), fusedcycloalkyl, fused cycloalkenyl, fused heterocycle, or fused aryl, wherethe aforementioned cycloalkyl, cycloalkenyl, heterocycle and arylsubstituents can themselves be optionally substituted.

The term “aryl” refers to cyclic, aromatic hydrocarbon groups that have1 to 5 aromatic rings, especially monocyclic or bicyclic groups such asphenyl, biphenyl or naphthyl. Where containing two or more aromaticrings (bicyclic, etc.), the aromatic rings of the aryl group may bejoined at a single point (e.g., biphenyl), or fused (e.g., naphthyl,phenanthrenyl and the like). “Substituted aryl” refers to an aryl groupsubstituted by one or more substituents, preferably 1 to 3 substituents,at any point of attachment. Exemplary substituents include, but are notlimited to, nitro, cycloalkyl or substituted cycloalkyl, cycloalkenyl orsubstituted cycloalkenyl, cyano, alkyl or substituted alkyl, as well asthose groups recited above as exemplary alkyl substituents. Theexemplary substitutents can themselves be optionally substituted.Exemplary substituents also include fused cylic groups, especially fusedcycloalkyl, fused cycloalkenyl, fused heterocycle, or fused aryl, wherethe aforementioned cycloalkyl, cycloalkenyl, heterocycle and arylsubstituents can themselves be optionally substituted.

The terms “heterocycle” and “heterocyclic” refer to fully saturated, orpartially or fully unsaturated, including aromatic (i.e., “heteroaryl”)cyclic groups (for example, 4 to 7 membered monocyclic, 7 to 11 memberedbicyclic, or 8 to 16 membered tricyclic ring systems) which have atleast one heteroatom in at least one carbon atom-containing ring. Eachring of the heterocyclic group containing a heteroatom may have 1, 2, 3,or 4 heteroatoms selected from nitrogen atoms, oxygen atoms and/orsulfur atoms, where the nitrogen and sulfur heteroatoms may optionallybe oxidized and the nitrogen heteroatoms may optionally be quatemized.(The term “heteroarylium” refers to a heteroaryl group bearing aquaternary nitrogen atom and thus a positive charge.) The heterocyclicgroup may be attached to the remainder of the molecule at any heteroatomor carbon atom of the ring or ring system. Exemplary monocyclicheterocyclic groups include azetidinyl, pyrrolidinyl, pyrrolyl,pyrazolyl, oxetanyl, pyrazolinyl, imidazolyl, imidazolinyl,imidazolidinyl, oxazolyl, oxazolidinyl, isoxazolinyl, isoxazolyl,thiazolyl, thiadiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl,furyl, tetrahydrofuryl, thienyl, oxadiazolyl, piperidinyl, piperazinyl,2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl,azepinyl, hexahydrodiazepinyl, 4-piperidonyl, pyridyl, pyrazinyl,pyrimidinyl, pyridazinyl, triazinyl, triazolyl, tetrazolyl,tetrahydropyranyl, moutholinyl, thiamorpholinyl, thiamorpholinylsulfoxide, thiamorpholinyl sulfone, 1,3-dioxolane andtetrahydro-1,1-dioxothienyl, and the like. Exemplary bicyclicheterocyclic groups include indolyl, isoindolyl, benzothiazolyl,benzoxazolyl, benzoxadiazolyl, benzothienyl, benzo[d][1,3]dioxolyl,2,3-dihydrobenzo[b][1,4]dioxinyl, quinuclidinyl, quinolinyl,tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl,indolizinyl, benzofuryl, benzofurazanyl, chromonyl, coumarinyl,benzopyranyl, cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridyl,furopyridinyl (such as furo[2,3-c]pyridinyl, furo[3,2-b]pyridinyl] orfuro[2,3-b]pyridinyl), dihydroisoindolyl, dihydroquinazolinyl (such as3,4-dihydro-4-oxo-quinazolinyl), triazinylazepinyl, tetrahydroquinolinyland the like. Exemplary tricyclic heterocyclic groups includecarbazolyl, benzidolyl, phenanthrolinyl, acridinyl, phenanthridinyl,xanthenyl and the like. Thiazole?

“Substituted heterocycle” and “substituted heterocyclic” (such as“substituted heteroaryl”) refer to heterocycle or heterocyclic groupssubstituted with one or more substituents, preferably 1 to 4substituents, at any available point of attachment. Exemplarysubstituents include, but are not limited to, cycloalkyl or substitutedcycloalkyl, cycloalkenyl or substituted cycloalkenyl, nitro, oxo (i.e.,═O), cyano, alkyl or substituted alkyl, as well as those groups recitedabove as exemplary alkyl substituents. The exemplary substitutents canthemselves be optionally substituted. Exemplary substituents alsoinclude spiro-attached or fused cylic substituents at any availablepoint or points of attachment, especially spiro-attached cycloalkyl,spiro-attached cycloalkenyl, Spiro-attached heterocycle (excludingheteroaryl), fused cycloalkyl, fused cycloalkenyl, fused heterocycle, orfused aryl, where the aforementioned cycloalkyl, cycloalkenyl,heterocycle and aryl substituents can themselves be optionallysubstituted.

The terms “halogen” or “halo” refer to chlorine, bromine, fluorine oriodine.

The term “carbocyclic” refers to aromatic or non-aromatic 3 to 7membered monocyclic and 7 to 11 membered bicyclic groups, in which allatoms of the ring or rings are carbon atoms. “Substituted carbocyclic”refers to a carbocyclic group substituted with one or more substituents,preferably 1 to 4 substituents, at any available point of attachment.Exemplary substituents include, but are not limited to, nitro, cyano,OR_(a), wherein R_(a) is as defined hereinabove, as well as those groupsrecited above as exemplary cycloalkyl substituents. The exemplarysubstitutents can themselves be optionally substituted.

The term “pharmaceutically-acceptable excipient, carrier, or diluent” asused herein means a pharmaceutically-acceptable material, composition orvehicle, such as a liquid or solid filler, diluent, excipient, solventor encapsulating material, involved in carrying or transporting thesubject pharmaceutical agent from one organ, or portion of the body, toanother organ, or portion of the body. Each carrier must be “acceptable”in the sense of being compatible with the other ingredients of theformulation and not injurious to the patient. Some examples of materialswhich can serve as pharmaceutically-acceptable carriers include: sugars,such as lactose, glucose and sucrose; starches, such as corn starch andpotato starch; cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatin; talc; excipients, such as cocoa butter andsuppository waxes; oils, such as peanut oil, cottonseed oil, saffloweroil, sesame oil, olive oil, corn oil and soybean oil; glycols, such aspropylene glycol; polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol; phosphate buffer solutions; and other non-toxiccompatible substances employed in pharmaceutical formulations. Wettingagents, emulsifiers and lubricants, such as sodium lauryl sulfate,magnesium stearate, and polyethylene oxide-polypropylene oxide copolymeras well as coloring agents, release agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the compositions.

Unless otherwise indicated, any heteroatom with unsatisfied valences isassumed to have hydrogen atoms sufficient to satisfy the valences.

The compounds of the present invention may form salts which are alsowithin the scope of this invention. Reference to a compound of thepresent invention herein is understood to include reference to saltsthereof, unless otherwise indicated. The term “salt(s)”, as employedherein, denotes acidic and/or basic salts formed with inorganic and/ororganic acids and bases. In addition, when a compound of the presentinvention contains both a basic moiety, such as but not limited to apyridine or imidazole, and an acidic moiety such as but not limited to acarboxylic acid, zwitterions (“inner salts”) may be formed and areincluded within the term “salt(s)” as used herein. Pharmaceuticallyacceptable (i.e., non-toxic, physiologically acceptable) salts arepreferred, although other salts are also useful, e.g., in isolation orpurification steps which may be employed during preparation. Salts ofthe compounds of the present invention may be formed, for example, byreacting a compound I, II or III with an amount of acid or base, such asan equivalent amount, in a medium such as one in which the saltprecipitates or in an aqueous medium followed by lyophilization.

The compounds of the present invention which contain a basic moiety,such as but not limited to an amine or a pyridine or imidazole ring, mayform salts with a variety of organic and inorganic acids. Exemplary acidaddition salts include acetates (such as those formed with acetic acidor trihaloacetic acid, for example, trifluoroacetic acid), adipates,alginates, ascorbates, aspartates, benzoates, benzenesulfonates,bisulfates, borates, butyrates, citrates, camphorates,camphorsulfonates, cyclopentanepropionates, digluconates,dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates,glycerophosphates hemisulfates, heptanoates, hexanoates, hydrochlorides,hydrobromides, hydroiodides, hydroxyethanesulfonates (e.g.,2-hydroxyethanesulfonates), lactates, maleates, methanesulfonates,naphthalenesulfonates (e.g., 2-naphthalenesulfonates), nicotinates,nitrates, oxalates, pectinates, persulfates, phenylpropionates (e.g.,3-phenylpropionates), phosphates, picrates, pivalates, propionates,salicylates, succinates, sulfates (such as those formed with sulfuricacid), sulfonates, tartrates, thiocyanates, toluenesulfonates such astosylates, undecanoates, and the like.

The compounds of the present invention which contain an acidic moiety,such but not limited to a carboxylic acid, may form salts with a varietyof organic and inorganic bases. Exemplary basic salts include ammoniumsalts, alkali metal salts such as sodium, lithium and potassium salts,alkaline earth metal salts such as calcium and magnesium salts, saltswith organic bases (for example, organic amines) such as benzathines,dicyclohexylamines, hydrabamines (formed with N,N-bis(dehydroabietyl)ethylenediamine), N-methyl-D-glucamines, N-methyl-D-glycamides, t-butylamines, and salts with amino acids such as arginine, lysine and thelike. Basic nitrogen-containing groups may be quaternized with agentssuch as lower alkyl halides (e.g. methyl, ethyl, propyl, and butylchlorides, bromides and iodides), dialkyll sulfates (e.g. dimethyl,diethyl, dibutyl, and diamyl sulfates), long chain halides (e.g. decyl,lauryl, myristyl and stearyl chlorides, bromides and iodides), aralkylhalides (e.g. benzyl and phenethyl bromides), and others.

Solvates of the compounds of the invention are also contemplated herein.Solvates of the compounds of the present invention include, for example,hydrates.

Compounds of the present invention, and salts thereof, may exist intheir tautomeric form (for example, as an amide or imino ether). Allsuch tautomeric forms are contemplated herein as part of the presentinvention.

All stereoisomers of the compounds of the present invention (forexample, those which may exist due to asymmetric carbons on varioussubstituents), including enantiomeric forms and diastereomeric forms,are contemplated within the scope of this invention. Individualstereoisomers of the compounds of the invention may, for example, besubstantially free of other isomers (e.g., as a pure or substantiallypure optical isomer having a specified activity), or may be admixed, forexample, as racemates or with all other, or other selected,stereoisomers. The chiral centers of the present invention may have theS or R configuration as defined by the IUPAC 1974 Recommendations. Theracemic forms can be resolved by physical methods, such as, for example,fractional crystallization, separation or crystallization ofdiastereomeric derivatives or separation by chiral columnchromatography. The individual optical isomers can be obtained from theracemates by any suitable method, including without limitation,conventional methods, such as, for example, salt formation with anoptically active acid followed by crystallization.

Compounds of the present invention are, subsequent to their preparation,preferably isolated and purified to obtain a composition containing anamount by weight equal to or greater than 99% (“substantially pure”compound I), which is then used or formulated as described herein.

All configurational isomers of the compounds of the present inventionare contemplated, either in admixture or in pure or substantially pureform. The definition of compounds of the present invention embraces bothcis (Z) and trans (E) alkene isomers, as well as cis and trans isomersof cyclic hydrocarbon or heterocyclic rings.

Throughout the specifications, groups and substituents thereof may bechosen to provide stable moieties and compounds.

B. Compounds

In one aspect, the present invention provides a compound of formula I,

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptablesalt or solvate thereof, wherein the symbols have the following meaningsand are, for each occurrence, independently selected:

-   X is O or S;-   R₁ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkynyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a);-   R₃ is hydrogen, cyano, OCF₃, alkyl or substituted alkyl, alkenyl or    substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or    substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl,    heterocycle or substituted heterocycle, aryl or substituted aryl,    OR_(a), SR_(a), or NR_(b)R_(c);-   R₇ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkynyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a);-   R_(a) is hydrogen, alkyl or substituted alkyl, alkenyl or    substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or    substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl,    heterocycle or substituted heterocycle, or aryl or substituted aryl;-   R_(b) and R_(c) are independently hydrogen, alkyl or substituted    alkyl, cycloalkyl or substituted cycloalkyl, heterocycle or    substituted heterocycle, or aryl or substituted aryl, or said R_(b)    and R_(c) together with the N to which they are bonded optionally    form a heterocycle or substituted heterocycle; and-   n is 1-4,-   provided that when R₃ is not NR_(b)R_(c), then R₇ is not hydrogen    and at least one of R₁ and R₇ is halogen, aryl, or substituted aryl.

In certain embodiments, the present invention provides a compoundselected from the group consisting of:

In certain other embodiments, the present invention provides a compoundselected from the group consisting of:

In another aspect, the present invention provides a compound of formulaII,

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptablesalt or solvate thereof, wherein the symbols have the following meaningsand are, for each occurrence, independently selected:

-   X is O or S;-   R₁ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkenyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, or SR_(a);-   R₄ is hydrogen, alkyl or substituted alkyl, alkenyl or substituted    alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted    cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle or    substituted heterocycle, aryl or substituted aryl, alkylaryl or    substituted alkylaryl;-   R₅ is hydrogen, alkyl or substituted alkyl, cycloalkyl or    substituted cycloalkyl, aryl or substituted aryl, alkylaryl or    substituted alkylaryl; optionally, R₄ and R₅ may be combined to form    alkenyl or substituted alkenyl;-   R₇ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkynyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a);-   R_(a), is hydrogen, alkyl or substituted alkyl, alkenyl or    substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or    substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl,    heterocycle or substituted heterocycle, or aryl or substituted aryl;    and-   n is 1-4,-   provided that at least one of R₁ and R₇ is halogen; or at least one    of R₁, R₄, R₅ and R₇ is aryl or substituted aryl.

In certain embodiments, the compound of formula II is selected from thegroup consisting of:

In yet another aspect, the present invention provides a compound offormula III,

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptablesalt or solvate thereof, wherein the symbols have the following meaningsand are, for each occurrence, independently selected:

-   X is O or S;-   R₁ is halogen;-   R₄ is hydrogen, alkyl or substituted alkyl, alkenyl or substituted    alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted    cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle or    substituted heterocycle, aryl or substituted aryl, alkylaryl or    substituted alkylaryl;-   R₅ is hydrogen, alkyl or substituted alkyl, cycloalkyl or    substituted cycloalkyl, aryl or substituted aryl, alkylaryl or    substituted alkylaryl; optionally, R₄ and R₅ may be combined to form    alkenyl or substituted alkenyl;-   R₆ is hydrogen, alkyl or substituted alkyl, OR_(a), OC(═O)R_(a), or    SR_(a);-   R₇ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkynyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a);-   R_(a) is hydrogen, alkyl or substituted alkyl, alkenyl or    substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or    substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl,    heterocycle or substituted heterocycle, or aryl or substituted aryl;    and-   n is 1-4.

In certain embodiments, the compound of formula III is

In yet another aspect, the present invention provides a compound offormula IV,

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptablesalt or solvate thereof, wherein the symbols have the following meaningsand are, for each occurrence, independently selected:

-   X is O or S;-   R₁ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkynyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a);-   R₇ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkynyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a);-   R₈ is hydrogen, alkyl or substituted alkyl, cycloalkyl or    substituted cycloalkyl, aryl or substituted aryl, alkylaryl or    substituted alkylaryl;-   R₉ and R₁₀ are each independently hydrogen, alkyl or substituted    alkyl, cycloalkyl or substituted cycloalkyl, aryl or substituted    aryl, heterocycle or substituted heterocycle, alkylaryl or    substituted alkylaryl, alkylheteroaryl or substituted    alkylheteroaryl; or R₉ and R₁₀ together with the carbon to which    they are bonded optionally form cycloalkyl or substituted    cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle or    substituted heterocycle;-   R_(a), is hydrogen, alkyl or substituted alkyl, alkenyl or    substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or    substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl,    heterocycle or substituted heterocycle, or aryl or substituted aryl;    and-   n is 1-4.

In certain embodiments, the compound of formula IV is selected from thegroup consisting of:

In yet another aspect, the present invention provides a compound offormula V,

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptablesalt or solvate thereof, wherein the symbols have the following meaningsand are, for each occurrence, independently selected:

-   X is O or S;-   R₁ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkynyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a);-   R₇ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkynyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a),-   R₁₁ is hydrogen, alkyl or substituted alkyl, alkenyl or substituted    alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted    cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle or    substituted heterocycle, aryl or substituted aryl, alkylaryl or    substituted alkylaryl, alkylheteroaryl or substituted alkyl    heteroaryl;-   R_(a) is hydrogen, alkyl or substituted alkyl, alkenyl or    substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or    substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl,    heterocycle or substituted heterocycle, or aryl or substituted aryl;    and-   n is 1-4.-   In yet another aspect, the present invention provides a compound of    formula.

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptablesalt or solvate thereof, wherein the symbols have the following meaningsand are, for each occurrence, independently selected:

-   X is O or S;-   each R₁ is independently hydrogen, halogen, cyano, nitro, CF₃, OCF₃,    alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkynyl    or substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a);-   R₃ is hydrogen, cyano, CF₃, OCF₃, alkyl or substituted alkyl,    alkenyl or substituted alkenyl, alkynyl or substituted alkynyl,    cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted    cycloalkenyl, heterocycle or substituted heterocycle, aryl or    substituted aryl, OR_(a), SR_(a), or NR_(b)R_(c);-   R₇ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkynyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a);-   R_(a) is hydrogen, alkyl or substituted alkyl, alkenyl or    substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or    substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl,    heterocycle or substituted heterocycle, or aryl or substituted aryl;-   R_(b) and R_(c) are independently hydrogen, alkyl or substituted    alkyl, cycloalkyl or substituted cycloalkyl, heterocycle or    substituted heterocycle, or aryl or substituted aryl, or said R_(b)    and R_(c) together with the N to which they are bonded optionally    form a heterocycle or substituted heterocycle; and-   provided that when R₃ is hydroxyl, alkyl, or substituted alkyl, then    R₇ is halogen, aryl, or substituted aryl;-   further provided that when R is aryl or substituted aryl, then R₇ is    not hydrogen, and-   further provided that    2-acetyl-7-chloro-naphtho[2,3-b]furan-4,9-dione and    2-acetyl-7-fluoro-naphtho[2,3-b]furan-4,9-dione are excluded.

In certain embodiments, the present invention provides a compoundselected from the group consisting of:

In yet another aspect, the present invention provides a compound offormular VII:

-   R₁ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkynyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a);-   R₃ is hydrogen, cyano, OCF₃, alkyl or substituted alkyl, alkenyl or    substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or    substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl,    heterocycle or substituted heterocycle, aryl or substituted aryl,    OR_(a), SR_(a), or NR_(b)R_(c);-   R₇ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkynyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a);-   R_(a) is hydrogen, alkyl or substituted alkyl, alkenyl or    substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or    substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl,    heterocycle or substituted heterocycle, or aryl or substituted aryl;-   R_(b) and R_(c) are independently hydrogen, alkyl or substituted    alkyl, cycloalkyl or substituted cycloalkyl, heterocycle or    substituted heterocycle, or aryl or substituted aryl, or said R_(b)    and R_(c) together with the N to which they are bonded optionally    form a heterocycle or substituted heterocycle; and-   n is 1-4,-   provided that when R₃ is not NR_(b)R_(c) then R₇ is not hydrogen.

In yet another aspect, the present invention provides a compound offormula VIII:

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptablesalt or solvate thereof, wherein the symbols have the following meaningsand are, for each occurrence, independently selected:

-   X is O or S;-   R₁ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkynyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a);-   R₇ is hydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or    substituted alkyl, alkenyl or substituted alkenyl, alkynyl or    substituted alkynyl, cycloalkyl or substituted cycloalkyl,    cycloalkenyl or substituted cycloalkenyl, heterocycle or substituted    heterocycle, aryl or substituted aryl, OR_(a), or SR_(a);-   R₁₂ is hydrogen, alkyl or substituted alkyl, alkenyl or substituted    alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted    cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle or    substituted heterocycle, aryl or substituted aryl, alkylaryl or    substituted alkylaryl, —C(═O)R₃, or —C(OH)R₄R₅;-   R₃ is hydrogen, cyano, CF₃, OCF₃, alkyl or substituted alkyl,    alkenyl or substituted alkenyl, alkynyl or substituted alkynyl,    cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted    cycloalkenyl, heterocycle or substituted heterocycle, aryl or    substituted aryl, OR_(a), SR_(a), or NR_(b)R_(c);-   R₄ is hydrogen, alkyl or substituted alkyl, alkenyl or substituted    alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted    cycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle or    substituted heterocycle, aryl or substituted aryl, alkylaryl or    substituted alkylaryl;-   R₅ is hydrogen, alkyl or substituted alkyl, cycloalkyl or    substituted cycloalkyl, aryl or substituted aryl, alkylaryl or    substituted alkylaryl, optionally, R₄ and R₅ may be combined to form    alkenyl or substituted alkenyl;-   R_(a) is hydrogen, alkyl or substituted alkyl, alkenyl or    substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or    substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl,    heterocycle or substituted heterocycle, or aryl or substituted aryl;-   R_(b) and R_(c) are independently hydrogen, alkyl or substituted    alkyl, cycloalkyl or substituted cycloalkyl, heterocycle or    substituted heterocycle, or aryl or substituted aryl, or said R_(b)    and R_(c) together with the N to which they are bonded optionally    form a heterocycle or substituted heterocycle; and-   n is 1-4;-   provided that 2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione,    2-acetyl-7-chloro-naphtho[2,3-b]furan-4,9-dione,    2-acetyl-7-fluoro-naphtho[2,3-b]furan-4,9-dione,    2-acetylnaphtho[2,3-b]furan-4,9-dione, and    2-ethyl-naphtho[2,3-b]furan-4,9-dione are excluded.

In certain embodiments, the compound of formula VIII is selected fromthe group consisting of:

C. Uses

Stat3 pathway can be activated in response to cytokines, such as IL-6,or by a series of tyrosine kinases, such as EGFR, JAKs, Abl, KDR, c-Met,Src, and Her2. The downstream effectors of Stat3 include but not limitedto Bcl-x1, c-Myc, cyclinD1, Vegf, MMP-2, and survivin. Stat3 pathway isfound to be aberrantly active in a wide variety of human diseases, asshown in Table 1. Existing clinical samples examined showed thatpersistently active Stat3 pathway occurs in more than half of breast andlung cancers, hepatocellular carcinomas, multiple myelomas and more than95% of head and neck cancers. Blocking Stat3 pathway causes cancercell-growth arrest, apoptosis, and reduction of metastasis frequency invitro and/or in vivo. Activated Stat3 has also been demonstrated in anumber of autoimmune and inflammatory diseases. Furthermore, asinterleukin-6 mediated inflammation is the common causative origin forAtherosclerosis [34], Peripheral Vascular Disease [35, 36], CoronaryArtery Disease [35, 36], hypertension [37], Osteroprorosis [38], Type 2Diabetes [35], and Dementia [39] and gp130-Jaks-Stats is the mainpathway activated by IL-6, inhibition of the Stat3 pathway may treat orprevent these diseases as well. Therefore, Stat3 inhibitors are highlysought after therapeutic agents.

TABLE 1 Activation of STAT3 PATHWAY in human diseases DISEASES REF.ONCOLOGY Solid Breast Cancer [40] DISEASES Tumors Head and Neck Cancer(SCCHN) [41] Lung Cancer [42] Ovarian Cancer [43] Pancreatic Cancer [44]Colorectal carcinoma [45] Prostate Cancer [46] Renal Cell carcinoma [47]Melanoma [48] Hepatocellular carcinomas [12] Cervical Cancer [49]Endometrial Cancer [49] Sarcomas [50, 51] Brain Tumors [52] GastricCancers  [5] Hematologic Multiple Myeloma [53] Tumors LeukemiaHTLV-1-dependent Leukemia [54] Chronic Myelogenous Leukemia [47] AcuteMyelogenous Leukemia [55] Large Granular Lymphocyte Leukemia [56]Lymphomas EBV-related/Burkitt's [57] Mycosis Fungoides [47] HSVSaimiri-dependent (T-cell) [47] Cutaneous T-cell Lymphoma [58] Hodgkin'sDiseases [47] Anaplastic Large-cell Lymphoma [59] IMMUNE InflammatoryInflammatory Bowel Diseases [60] DISEASES Diseases InflammatoryArthritis [61-63] Crohn's Diseases [64] Chronic inflammatory conditions[65] Autoimmune Reumatoid Arthritis [61, 62, 66-68] Systemic lupuserythematosus [69] Asthma [70] Allergy [71] Infections [72]PROLIFERATIVE Psoriasis [73] DISORDERS Keloids [74] Warts [75]Myelodysplastic syndrome [76] Polycythemia vera [77] CNS Alzhemer's[78-80] DISEASES Multiple sclerosis (MS) [78, 80, 81]

The present invention provides, in part, Stat3 inibitors, comprising ofa compound of formula I-VIII of the present invention, or an enantiomer,diastereomer, tautomer, or pharmaceutically acceptable salt or solvatethereof.

The present invention further provides a method of treating a disorderrelated to aberrant Stat3 pathway activity in a mammal. The method oftreating the disorder comprises administering to the mammal in needthereof an amount of a compound of formulae I through VIII, or anenantiomer, diastereomer, tautomer, or pharmaceutically acceptable saltor solvate thereof. The said aberrant Stat3 pathway activity can beidentified by expression of phosphorylated Stat3 or its surrogateupstream or downstream regulators. In one embodiment, the condition is acancer related to aberrant Stat3 pathway activity. In anotherembodiment, the condition is an autoimmune or inflammatory diseaserelated to aberrant Stat3 pathway activity. The said autoimmune orinflammatory disease is selected from the group consisting ofinflammatory bowel diseases, arthritis, Crohn's diseases, ulcerativecolitis, rheumatoid arthritis, asthma, allergy, and systemic lupuserythematosus. In another embodiment, the condition is a CNS diseaserelated to aberrant Stat3 pathway activity. The said. CNS disease isselected from autoimmune demyelination disorder, Alzheimer's, stroke,ischemia reperfusion injury and multiple sclerosis. In yet anotherembodiment, the condition is a disease caused by inflammation andrelated to aberrant Stat3 pathway activity. These diseases includeatherosclerosis, peripheral vascular disease, coronary artery disease,hypertension, osteroprorosis, type 2 diabetes, or dementia.

Recent studies have uncovered the presence of cancer stem cells with anexclusive ability to regenerate tumors. These cancer stem cells arefunctionally linked with continued malignant growth, cancer metastasis,recurrence, and cancer drug resistance. Cancer stem cells and theirdifferentiated progeny appear to have markedly different biologiccharacteristics. They persist in tumors as a distinct, but rarepopulation. Conventional cancer drug screenings depend on measurement ofthe amount of tumor mass and, therefore, are unlikely to identify drugsthat act specifically on the stem cells. In fact, cancer stem cells havebeen demonstrated to be resistant to standard chemotherapies and areenriched after standard chemotherapy treatments, which result in cancerrefractory and recurrence. Cancer stem cells have also been demonstratedto be resistant to radiotherapy [17]. The reported cancer types in whichcancer stem cells have been isolated include breast cancer, head andneck cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectalcarcinoma, prostate cancer, melanoma, multiple myeloma, kaposi sarcoma,ewing's sarcoma, liver cancer, medulloblastoma, brain tumors, andleukemia. The mounting evidence linking cancer stem cells totumorigenesis provides enormous therapeutic opportunity for targetingcancer stem cells. The key to unlocking this untapped potential is theidentification and validation of pathways that are selectively importantfor cancer stem cell self-renewal and survival. Though multiple pathwaysunderlying tumorigenesis in cancer and in embryonic stem cells or adultstem cells have been elucidated in the past, no pathways have beenreported for cancer stem cell self-renewal and survival, largely due tothe absence of a good system for doing so. We have identified that Stat3is a key cancer stem cell survival and self-renewal factor. Therefore,Stat3 inhibitors can kill cancer stem cells and inhibit cancer stem cellself-renewal.

According to one or more embodiments of the present invention, cancerstem cell (CSC) or cancer stem cells (CSCs) refer to a minute populationof cancer cells that have self-renewal capability and are tumorigenic.They are also called “Cancer Initiating Cells”, “Tumor initiatingCells”, “Cancer Stem-Like Cells”, “Stem-Like Cancer Cells”, and “supermalignant cells”, etc. The methods of isolating these cells include butnot limited to identification by their ability of efflux Hoechst 33342,identification by the surface markers these cells expressed, such asCD133, CD44, CD166, and others, and enrichment by their tumorigenicproperty.

The present invention provides, in part, a method ofinhibiting/reducing/diminishing cancer stem cell survival and orself-renewal with an effective amount of a compound of formulae Ithrough VIII, or an enantiomer, diastereomer, tautomer, orpharmaceutically acceptable salt or solvate thereof. These cancer stemcells can be identified by the surface markers, such as CD44, CD133, andCD166.

As cancer stem cells are resistant to conventional chemotherapies, thepresent invention provides, in part, a method of treating cancerrefractory to conventional chemotherapies in a mammal, comprising to themammal in need thereof a pharmaceutical composition comprising acompound of formulae I through VIII, or pharmaceutically acceptable saltor solvate thereof.

As cancer stem cells are the root of cancer and are fundamentallyresponsible for cancer recurrence, the present invention provides, inpart, a method of treating recurrent cancer in a mammal that has failedsurgery, chemo, or XRT, comprising administering to the mammal in needthereof a pharmaceutical composition comprising a compound of formulae Ithrough VIII, or pharmaceutically acceptable salt or solvate thereof.

Similarly, as cancer stem cells are the seeds of cancer and arefundamentally responsible for cancer metastasis, the present inventionprovides, in part, a method of treating or preventing cancer metastasisin a mammal, comprising administering to the mammal in need thereof apharmaceutical composition comprising a compound of formulae I throughVIII, or pharmaceutically acceptable salt or solvate thereof.

The present invention further provides, in part, a method of treatingcancer in a mammal, comprising administering to the mammal atherapeutically effective amount of a compound of formula I-VIII of thepresent invention, or an enantiomer, diastereomer, tautomer, orpharmaceutically acceptable salt or solvate thereof. In one embodiment,the said cancer above is selected from lung cancer, breast cancer,cervix cancer, colon cancer, liver cancer, pancreatic cancer, head andneck cancer, gastric cancer, and prostate cancer.

The present invention further provides, in part, a pharmaceuticalcomposition comprising a compound of formulae I through VII, or anenantiomer, diastereomer, tautomer, or pharmaceutically acceptable saltor solvate thereof, and a pharmaceutically-acceptable excipient,carrier, or diluent.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the mammal being treated and the particular mode ofadministration. The amount of active ingredient, which can be combinedwith a carrier material to produce a single dosage form, will generallybe that amount of the compound which produces a therapeutic effect.Generally, out of 100%, this amount will range, for example, from about1% to about 99% of active ingredient, from about 5% to about 70%, fromabout 10% to about 30%.

Therapeutic compositions or formulations of the invention suitable fororal administration may be in the form of capsules, cachets, pills,tablets, lozenges (using a flavored basis, usually sucrose and acacia ortragacanth), powders, granules, or as a solution or a suspension in anaqueous or non-aqueous liquid, or as an oil-in-water or water-in-oilliquid emulsion, or as an elixir or syrup, or as pastilles (using aninert base, such as gelatin and glycerin, or sucrose and acacia) and/oras mouth washes and the like, each containing a predetermined amount ofa compound of the present invention as an active ingredient. A compoundof the present invention may also be administered as a bolus, electuaryor paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), thealcohol or inhibitor according to the invention is mixed with one ormore pharmaceutically-acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: fillers or extenders,such as starches, lactose, sucrose, glucose, mannitol, and/or silicicacid; binders, such as, for example, carboxymethylcellulose, alginates,gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, suchas glycerol; disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,sodium carbonate, and sodium starch glycolate; solution retardingagents, such as paraffin; absorption accelerators, such as quaternaryammonium compounds; wetting agents, such as, for example, cetyl alcohol,glycerol monostearate, and polyethylene oxide-polypropylene oxidecopolymer; absorbents, such as kaolin and bentonite clay; lubricants,such a talc, calcium stearate, magnesium stearate, solid polyethyleneglycols, sodium lauryl sulfate, and mixtures thereof; and coloringagents. In the case of capsules, tablets and pills, the pharmaceuticalcompositions may also comprise buffering agents. Solid compositions of asimilar type may also be employed as fillers in soft and hard-filledgelatin capsules using such excipients as lactose or milk sugars, aswell as high molecular weight polyethylene glycols and the like.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof. Additionally, cyclodextrins,e.g., hydroxypropyl-.beta.-cyclodextrin may be used to solubilizecompounds.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents. Suspensions, inaddition to the alcohols or inhibitors according to the invention, maycontain suspending agents as, for example, ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more alcohols or inhibitorsaccording to the invention, with one or more suitable nonirritatingexcipients or carriers comprising, for example, cocoa butter,polyethylene glycol, a suppository wax or a salicylate, and which issolid at room temperature, but liquid at body temperature and,therefore, will melt in the rectum or vaginal cavity and release theactive pharmaceutical agents of the invention. Formulations of thepresent invention which are suitable for vaginal administration alsoinclude pessaries, tampons, creams, gels, pastes, foams or sprayformulations containing such carriers as are known in the art to beappropriate.

Dosage forms for the topical or transdermal administration of an alcoholor other inhibitor according to the invention include powders, sprays,ointments, pastes, creams, lotions, gels, solutions, patches andinhalants. The active compound may be mixed under sterile conditionswith a pharmaceutically-acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to analcohol or other inhibitor according to the invention, excipients, suchas animal and vegetable fats, oils, waxes, paraffins, starch,tragacanth, cellulose derivatives, polyethylene glycols, silicones,bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more alcohols or inhibitors according tothe invention in combination with one or morepharmaceutically-acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

In some cases, in order to prolong the effect of the alcohol orinhibitor according to the invention, it is desirable to slow theabsorption of the alcohol or inhibitor from subcutaneous orintramuscular injection. This may be accomplished by the use of a liquidsuspension of crystalline or amorphous material having poor watersolubility. The rate of absorption of the drug then depends upon itsrate of dissolution, which, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of aparenterally-administered composition is accomplished by dissolving orsuspending the alcohol or inhibitor in an oil vehicle. One strategy fordepot injections includes the use of polyethylene oxide-polypropyleneoxide copolymers wherein the vehicle is fluid at room temperature andsolidifies at body temperature.

The pharmaceutical compounds of this invention may be administered aloneor in combination with other pharmaceutical agents, or with otheranti-cancer therapies as described hereinabove, as well as incombination with a pharmaceutically-acceptable excipient, carrier, ordiluent as described above.

D. Chemical Synthesis

The compounds of the present invention can be prepared using the methodsdescribed below, together with synthetic methods known one skilled inthe art of organic synthesis, or variations thereon. The reactions areperformed in solvents appropriate to the reagents and materials employedand are suitable for transformations being effected. The startingmaterials for the examples contained herein are either commerciallyavailable or are readily prepared by standard methods from knownmaterials. For example, the following reactions are illustrations butnot limitations of the preparation of some of the starting materials andexamples used herein.

The process shown in Scheme 1 can be used for the preparation ofcompounds in Formula II, III and IV when R₇ is hydrogen and X is oxygenfrom starting material 1-1 which is commercially available when R₁ ishydrogen or can be readily made by one skilled in the art when R₁ ishalogen. The reaction of 2-hydroxy-1,4-naphthoquinone 1-1 with theappropriate aldehydes gives 2-hydroxy-3-(1-Alkenyl)-1,4-naphthoquinone1-2. Treatment of compound 1-2 with mercury acetate followed byhydrochloric acid affords 2-alkyl-naphtha[2,3-b]furan-4,9-dione 1-3.Oxidation of 1-3 with n-bromosuccinimide and lead tetraacetate gives2-(1-acetoxy-alkyl)-4H,9H-naphtho[2,3-b]furan-4,9-dione 1-4.Hydrochloric acid treatment of compound 1-4 gives 2-(hydroxy-alkyl orarylmethyl)-4H,9H-naphtho[2,3-b]furan-4,9-dione 1-5 and2-vinyl-4H,9H-naphtho[2,3-b]furan-4,9-dione 1-6.

The process shown in Scheme 2 can be used for the preparation ofcompounds in Formula III and IV when R₇ is not hydrogen and X is oxygenfrom same starting material 1-1 as shown in Scheme 1. The reaction of2-hydroxy-1,4-naphthoquinone 14 with the appropriate allylbromide gives2-allyloxy-1,4-naphthoquinone 2-2. Rearrangement of 2-2 in ethanolafford 2-hydroxy-3-allyl-1,4-naphthoquinone 2-3, which can be cyclizedby sulfuric acid treatment to form orthonaphthoquinone 2-4. Oxidation of2-4 with n-bromosticcinimide and lead tetraacetate gives3-acetoxy-orthonaphthoquinone 2-5. Hydrochloric acid treatment ofcompound 2-5 gives 2-alkyl (or aryl)-3-alkyl (oraryl)-naphtha[2,3-b]furan-4,9-dione 2-6.

The process shown in Scheme 3 can be used for the preparation of FormulaI compounds when X is O by using 1-3 (or 2-6) as starting material.Oxidation of 2-alkyl (or benzyl)-7-alkyl (or aryl,hydrogen)-naphtha[2,3-b]furan-4,9-dione 1-3 (or 2-6) with chromiumtrioxide yields compound of formula 3-2.

The process shown in Scheme 4 can be used for the preparation of FormulaI, VI and VII compounds.

The following non-limiting examples further illustrate the preparationof some of the starting materials and examples used herein.

EXAMPLE 1 Preparation of Sodium Salt of2-hydroxy-7-chloro-1,4-naphthoquinone 1-1 (R₁=Cl)

To the solution of 10 gram (0.06 mol) of 5-chloro-1-indanone in 200 mlof ethyl ether cooled in ice bath, 22 ml (0.066 mol) of 3 Mmethylmagnesium bromide in ethyl ether was dropped slowly over 30 min.The reaction mixture was stirred at room temperature overnight and thenevaporated to dryness. 150 ml of 2 N hydrochloric acid in 50% ethanolwas slowly dropped into the residue, and then refluxed for 1 hr. Themixture was extracted with dichloromethane and then the organic phasewas washed with water and dried with sodium sulfate. The intermediateproduct 3-methyl-6-chloro-indene was purified by silica gelchromatograph.

To a vigorously stirred solution of 18 gram of sodium dichromatehydrate, 1 gram of sodium benzene sulfonate, and 50 ml of sulfuric acidin 250 ml of water, at 55° C., 7.5 gram (0.046 mol) of3-methyl-6-chloro-indene was added dropwise in 1 hour. The mixture wasthen stirred for additional 20 minutes at 55° C. After chillingovernight at 0° C., the mixture was filtered, and the resulting solidwas washed successively with cold water and benzene and dried undervacuum.

The crude intermediate product (5-chloro-2-acetyl)phenylacetic acid wasdissolved in the mixture of 100 ml of anhydrous ethanol and 10 ml ofsulfuric acid. Then the resulting mixture was stirred for 48 hours atroom temperature. After diluting with 200 ml of water, the mixture wasextracted with dichloromethane, and then the organic phase was washedwith water and dried with sodium sulfate. The intermediate product ethyl(5-chloro-2-acetyl)phenylacetate was purified by silica gelchromatograph.

1.15 gram (0.050 mol) of sodium metal was suspended in 150 ml ofanhydrous ethanol with vigorously stirring. After the sodium metaldisappeared, 6 gram (0.025 mol) of ethyl(5-chloro-2-acetyl)phenylacetate was added, and the resulting mixturewas stirred at room temperature in an open flask for 24 hours. Themixture was chilled to 0° C. and filtered, and the resulting brick redsolid was washed with cold ethanol and dried under vacuum. 3.8 gram ofsodium salt of 2-hydroxy-7-chloro-1,4-naphthoquinone 1-1 (R₁=Cl) wasobtained: overall yield 27.5%. Mass (M-H) is 207.

EXAMPLE 2 Preparation of Sodium Salt of2-hydroxy-7-fluoro-1,4-naphthoquinone 1-1 (R₁=F)

Sodium salt of 2-hydroxy-7-fluoro-1,4-naphthoquinone (R₁=F) was obtainedfrom 10 gram (0.067 mol) of 5-fluoro-1-indanone by using the proceduredescribed in example 1 to give brick red solid: 30% yield. Mass (M-H) is191.

EXAMPLE 3 Preparation of 2-hydroxy-3-(1-n-butenyl)-1,4-naphthoquinone1-2 (R₁=H, R₄=CH₃)

To a solution of 20 gram (0.11 moles) of 2-hydroxy-1,4-naphthoquinone in150 ml of DIVISO and 20 ml of concentrated hydrochloride (37%) solutionat 75° C., 20 ml of n-butyraldehyde (0.23 mol) was added. The mixturewas vigorously stirred at the temperature of 72-78° C. for 4 hours, andthen cooled by addition of 300 ml of ice water, and the resultingmixture was extracted with 300 ml of dichloromethane twice. The combinedorganic phases was washed successively with 500 ml of water, and 500 mlof 5% sodium bisulfite, and 500 ml of 4% sodium bicarbonate, and finallyextracted with 400 ml of 5% sodium carbonate twice. The combined sodiumcarbonate extract was neutralized by addition of concentratedhydrochloric acid to pH 7.2-7.6. After chilling to 0° C., the mixturewas filtered, and the resulting brick red solid was washed with coldwater and dried under vacuum. 9.6 gram of product was obtained: 38.6%yield. ¹H NMR (in CDCl₃) δ 1.12 (t, J=8, 3H), 2.31 (m, 2H), 6.60-6.65(m, 1H), 7.07-7.15 (m, 1H), 7.66-7.77 (m, 3H), 8.06-8.15 (m, 2H).

EXAMPLE 4 Preparation of 2-ethyl-4H,9H-naphtho[2,3-b]furan-4,9-dione 1-3(R₁=H, R₄=CH₃)

A mixture of 9.6 gram (0.04 mol) of2-hydroxy-3-(1-n-butenyl)-1,4-naphthoquinone 1-2 (R₁=H, R₄=CH₃) and 18.8gram (0.094 mol) of mercury acetate in 300 ml of acetic acid was stirredat room temperature for 3 hours. The reaction mixture was filtered andthen the filtrate was evaporated to dryness. The residue was suspendedin 200 ml of concentrated hydrochloride (37%)/ethanol (1:2) and refixedfor 1 hour. After cooling down slowly to 0° C., the reaction mixture wasfiltered and the resulting solid product was washed with cooled 70%ethanol, and recrystallized in 70% ethanol to afford 5.1 gram of yellowcrystals: 53.1% yield. 2-ethyl-4H,9H-naphtho[2,3-b]furan-4,9-dione 1-3(R₁=H, R₄=CH₃), ¹H NMR (in CDCl₃) δ 1.36 (t, J=8, 3H), 2.85 (q, J=7,2H), 6.62 (s, 1H), 7.72-7.76 (m, 2H), 8.15-8.22 (m, 2H).

EXAMPLE 5 Preparation of 2-methyl-4H,9H-naphtho[2,3-b]furan-4,9-dione1-3 (R₁=H, R₄=H)

The intermediate product 2-hydroxy-3-(1-n-propenyl)-1,4-naphthoquinone1-2 (R₁=H, R₄=H) was prepared according to the procedure described inexample 3 by using 2-hydroxy-1,4-naphthoquinone 1-1 (R₁=H) andn-propionaldehyde as starting material.2-methyl-4H,9H-naphtho[2,3-b]furan-4,9-dione 1-3 (R₁=H, R₄=H) wasobtained from 10 gram (0.047 mol) of 1-2 (R₁=H, R₄=H) by using theprocedure described in example 4 to afford yellow crystals; 50% yield.2-methyl-4H,9H-naphtho[2,3-b]furan-4,9-dione 1-3 (R₁=H, R₄=H), ¹H NMR(in CDCl₃) δ 2.52 (s, 3H), 6.61 (s, 1H), 7.70-7.77 (m, 2H), 8.14-8.22(m, 2H);

EXAMPLE 6 Preparation of 2-benzyl-4H,9H-naptho[2,3-b]furan-4,9-dione 1-3(R₁=H, R₄=C₆H₅)

The intermediate product2-hydroxy-3-(3-phenyl-1-n-propenyl)-1,4-naphthoquinone 1-2 (R₁=H,R₄=C₆H₅) was prepared according to the procedure described in example 3by using 2-hydroxy-1,4-naphthoquinone 1-1 (R₁=H) and hydrocinnamaldehydeas starting material. 2-benzyl-4H,9H-naphtho[2,3-b]furan-4,9-dione 1-3(R₁=H, R₄=C₆H₅) was obtained from 10 gram (0.035 mol) of 1-2 (R₁=H,R₄=C₆H₅) by using the procedure described in example 4 to afford yellowcrystals; 50% yield. 2-benzyl-4H,9H-naphtho[2,3-b]furan-4,9-dione 1-3(R₁=H, R₄=C₆H₅), ¹H NMR (in CDCl₃) δ 4.14 (s, 2H), 6.56 (s, 1H),7.27-7.38 (m, 5H), 7.70-7.77 (m, 2H), 8.14-8.22 (m, 2H);

EXAMPLE 7 Preparation of2-ethyl-7-chloro-4H,9H-naphtho[2,3-b]furan-4,9-dione 1-3 (R₁=Cl, R₄=CH₃

The intermediate product2-hydroxy-3-(1-n-butenyl)-7-chloro-1,4-naphthoquinone 1-2 (R₁=Cl,R₄=CH₃) was prepared according to the procedure described in example 3by using sodium salt of 2-hydroxy-7-chloro-1,4-naphthoquinone 1-1(R₁=Cl) and n-butyraldehyde as starting material.2-ethyl-7-chloro-4H,9H-naphtho[2,3-b]furan-4,9-dione 1-3 (R₁=Cl, R₁=CH₃)was obtained from 2 gram (0.0077 mol) of 1-2 (R₁=Cl, R₄=CH₃) by usingthe procedure described in example 4 to afford yellow crystals; 30%yield. 2-ethyl-7-chloro-4H,9H-naphtho[2,3-b]furan-4,9-dione 1-3 (R₁=Cl,R₄=CH₃) ¹H NMR (in CDCl₃) δ 1.36 (t, J=8, 3H), 2.85 (q, J=7, 2H), 6.63(s, 1H), 7.67 (d, J=1H), 8.11 (d, J=8, 1H), 8.17 (s, 1H).

EXAMPLE 8 Preparation of2-ethyl-7-fluoro-4H,9H-naphtho[2,3-b]furan-4,9-dione 1-3 (R₁=F, R₄=CH₃)

The intermediate product2-hydroxy-3-(1-n-butenyl)-7-fluoro-1,4-naphthoquinone 1-2 (R₁=F, R₄=CH₃)was prepared according to the procedure described in example 3 by usingsodium salt of 2-hydroxy-7-fluoro-1,4-naphthoquinone 1-1 (R₁=F) andn-butyraldehyde as starting material.2-ethyl-7-fluoro-4H,9H-naphtho[2,3-b]furan-4,9-dione 1-3 (R₁=F, R₄=CH₃)was obtained from 2 gram (0.0082 mol) of 1-2 (R₁=F, R₄=CH₃) by using theprocedure described in example 4 to afford yellow crystals; 30% yield.2-ethyl-7-fluoro-4H,9H-naphtho[2,3-b]furan-4,9-dione 1-3 (R₁=F, R₄=CH₃)¹H NMR (in CDCl₃) δ 1.36 (t, J=8, 3H), 2.86 (q, J=7, 2H), 6.63 (s, 1H),7.35-7.40 (m, 1H), 7.85-7.88 (m, 1H), 8.18-8.22 (m, 1H).

EXAMPLE 9 Preparation of2-(1-acetoxyethyl)-4H,9H-naphthol[2,3-b]furan-4,9-dione 1-4 (R₁=H,R₄=CH₃.)

To a solution of 4.53 gram (0.02 mol) of2-ethyl-4H,9H-naphtho[2,3-b]furan-4,9-dione 1-3 (R₁=H, R₄=CH₃) in 200 mlof benzene, was added 7 g (0.04 mol) of N-bromosuccinimide and 7 g(0.016 mol)lead (IV) acetate. The mixture was refluxed for 24 hours, andthen poured into 2 volume of 5% sodium bicarbonate solution. Afterfiltration, the organic phase was separated and washed with water anddried with sodium sulfate and finally evaporated to dryness. The residuewas purified by silica gel column chromatograph to yield pale yellowpowder: 60% yield,2-(1-acetoxy-ethyl)-4H,9H-naphtho[2,3-b]furan-4,9-dione (R₁=H, R₄=CH₃)¹H NMR (in CDCl₃) δ 2.12 (d, J=7, 3H), 2.96 (s, 3H), 5.25 (q, J=7, 1H),6.86 (s, 1H), 7.72-7.79 (in, 2H), 8.17-8.24 (m, 2H).

EXAMPLE 10 Preparation of2-(1-Hydroxyethyl)-4H,9H-naphtho[2,3-b]furan-4,9-dione 1-5 (R₁=H,R₄=CH₃) and 2-vinyl-4H,9H-naphtho[2,3-b]furan-4,9-dione 1-6 (R₁=H, R₉=H)

A mixture of 2.84 gram (0.01 mol) of2-(1-acetoxyethyl)-4H,9H-naphtho[2,3-b]furan-4,9-dione 1-4 (R₁=H,R₄=CH₃) and 200 ml of 2N HCl in 70% ethanol was refluxed for 1 hour.After addition of 1 volume of ice water, the mixture was extracted withdichloromethane twice. The combined organic phase was washed with waterand dried with sodium sulfate, and then evaporated to dryness. Theresidue was purified by silica gel column chromatograph to yield twopale yellow fractions. The late eluted fraction: 35% yield,2-(1-hydroxyethyl)-4H,9H-naphtho[2,3-b]furan-4,9-dione 1-5 (R₁=H,R₄=CH₃) ¹H NMR (in CDCl₃) δ 1.66 (d, J=7, 3H), 2.26 (broad s, 1H), 5.05(q, J=7, 1H), 6.92 (s, 1H), 7.72-7.78 (m, 2H), 8.16-8.23 (m, 2H); Theearly eluted fraction: 43% yield,2-vinyl-4H,9H-naphtho[2,3-b]furan-4,9-dione 1-6 (R₁=H, R₉=H) ¹H NMR (inCDCl₃) δ 3.57 (q, J=7, 2H), 4.62 (q, J=7, 1H), 6.85 (s, 1H), 7.72-7.78(m, 2H), 8.17-8.24 (m, 2H).

EXAMPLE 11 Preparation of2-(1-Hydroxyethyl)-7-fluoro-4H,9H-naphtho[2,3-b]furan-4,9-dione 1-5(R₁=F, R₄=CH₃) and 2-vinyl-7-fluoro-4H,9H-naphtho[2,3-b]furan-4,9-dione1-6 (R₁=F, R₉=H)

2-(1-acetoxyethyl)-4H,9H-naphtho[2,3-b]furan-4,9-dione 1-4 (R₁=F,R₄=CH₃) was prepared according to the procedure described in example 9by using 2-ethyl-7-fluoro-4H,9H-naphtho[2,3-b]furan-4,9-dione 1-3 (R₁=F,R₄=CH₃) with yield of 55%.2-(1-Hydroxyethyl)-7-fluoro-4H,9H-naphtho[2,3-b]furan-4,9-dione 1-5(R₁=F, R₄=CH₃) and 2-vinyl-7-fluoro-4H,9H-naphtho[2,3-b]furan-4,9-dione1-6 (R₁=F, R₉=H) were prepared according to the procedure described inexample 10 with 35% yield for 1-5 (R₁=F, R₄=CH₃) ¹H NMR (in CDCl₃) δ1.66 (d, J=7, 3H), 2.20 (broad s, 1H), 5.05 (broad, 1H), 6.86 (s, 1H),7.37-7.43 (m, 1H), 7.85-7.89 (m, 1H), 8.19-8.24 (m, 1H); and with 40%yield for 1-6 (R₁=F, R₉=H) ¹H NMR (in CDCl₃) δ 3.58 (q, J=7, 2H), 4.61(q, J=7, 1H), 6.86 (s, 1H), 7.37-7.42 (m, 1H), 7.88 (q, J=6, 1H), 8.22(q, J=4, 1H).

EXAMPLE 12 Preparation of2-methyl-3-phenyl-4H,9H-naphtho[2,3-b]furan-4,9-dione 2-6 (R₁=H, R₄=H,R₇=C₆H₅)

To a solution of 20 gram (0.11 moles) of 2-hydroxy-1,4-naphthoquinone in200 ml of DMSO, 16.5 grams (0.11 moles) of sodium iodide, 15.3 nil (0.11moles) of triethylamine and 23.8 grams (0.12 moles) of cinnamyl bromidewere added. The mixture was vigorously stirred at 50° C. overnight, andthen cooled by addition of 400 ml of ice water, and the resultingmixture was extracted with 300 ml of toluene twice. The combined organicphase was washed successively with 500 ml of water, and 400 ml of 2Nsodium hydroxide twice, and 500 ml of water. The organic phase was driedwith sodium sulfate and evaporated to dryness. The crude product 2-2(R₁=H, R₄=H, R₇=C₆H₅) residue was dissolved in 200 ml of anhydrousethanol and refluxed for 3 hours. After evaporation, the residue wasdissolved in 200 ml of toluene, and extracted with 200 ml of 2N sodiumhydroxide twice. The combined extract was neutralized by addition ofconcentrated hydrochloric acid to pH 3-5, and extracted with 300 ml ofdichloromethane. The dichloromethane solution was washed with equalvolume of water, and dried with sodium sulfate, and then evaporated toyield crude lapachol analog 2-3 (R₁=H, R₄=H, R₇=C₆H₅). To 2 grams ofcrude 2-3 (R₁=H, R₇=C₆H₅), 20 ml of sulfuric acid was added, and theresulting mixture was placed at room temperature for 1 hour. Thesulfuric acid mixture was poured into 200 ml of water and extracted with200 ml of dichloromethane twice to give crude dunione analog 2-4 (R₁=H,R₄=H, R₇=C₆H₅) which was then purified with silica gel chromatograph.Treatment of 2-4 (R₁=H, R₄=H, R₇=C₆H₅) with N-bromosuccinimide and lead(IV) acetate was performed according to the procedure described inexample 9 to give crude 2-5 (R₁=H, R₄=H, R₇=C₆H₅). Without silica gelchromatograph, the crude 2-5 (R₁=H, R₄=H, R₇=C₆H₅) was directlydissolved in 200 ml ethanol/concentrated HCl (1:1) and refluxed for 1hour to give crude 2-6 (R₁=H, R₄H, R₇=C₆H₅) which was purified withsilica gel chromatograph. Overall yield 10%.2-methyl-3-phenyl-4H,9H-naphtho[2,3-b]furan-4,9-dione 2-6 (R₁=H, R₄=H,R₇=C₆H₅) ¹H NMR (in CDCl₃) δ 2.51 (s, 3H), 7.42-7.50 (m, 5H), 7.71-7.74(m, 2H), 8.10-8.13 (m, 1H), 8.21-8.23 (m, 1H).

EXAMPLE 13 Preparation of 2-Acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione3-2 (R₁=H, R₃=CH₃, R₇=H)

To a solution of 5.52 gram (0.02 mol) of2-ethyl-4H,9H-naphtho[2,3-b]furan-4,9-dione 1-3 (R₁=H, R₄=CH₃) in 100 mlof acetic acid and acetic anhydride (3:1), was added chromium (VI) oxide(6 g, 0.06 mol) in four portions at the interval of 30 minutes whilestirred vigorously. After additional 48 hours at room temperature, themixture was added one volume of water, and then chilled to 0° C. in icebath and filtered. The resulting solid was washed with cold water, driedunder vacuum, and recrystallized in ethyl acetate to give light yellowgreen crystal: 56% yield, 2-acetyl-4H,9H-naphtho[2,3-b]furan-4,9-dione3-2 (R₁H, R₃=CH₃, R₇=H) ¹H NMR (in CDCl₃) δ 2.67 (s, 3H), 7.61 (s,1H-3), 7.79-7.84 (m, 2H), 8.22-8.28 (m, 2H).

EXAMPLE 14 Preparation of 2-benzoyl-4H,9H-naphtho[2,3-b]furan-4,9-dione3-2 (R₁=H, R₃=C₆H₅, R₇=H)

To a solution of 5.76 gram (0.02 mol) of2-benzyl-4H,9H-naphtho[2,3-b]furan-4,9-dione 1-3 (R₁=H, R₄=C₆H₅) in 100ml of acetic acid and acetic anhydride (3:1), was added chromium (VI)oxide (6 g, 0.06 mol) in four portions at the interval of 30 minuteswhile stirred vigorously. After additional 48 hours at room temperature,the mixture was added two volume of water, and extracted withdichloromethane. The organic phase was washed with water and dried withsodium sulfate. After evaporation, the residue was subject to silica gelcolumn chromatograph purification. The light yellow green powder wasobtained, 45% yield, 2-benzoyl-4H,9H-naphtho[2,3-b]furan-4,9-dione 3-2(R₁=H, R₄=C₆H₅, R₇=H) ¹H NMR (in CDCl₃) δ 7.56-7.60 (m, 2H). 7.66-7.70(m, 1H), 7.71 (s, 1H-3), 7.80-7.84 (m, 2H), 8.10-8.13 (m, 2H), 8.24-8.30(m, 2H).

EXAMPLE 15 Preparation of2-benzoyl-7-promo-4H,9H-naphtho[2,3-b]furan-4,9-dione 3-2 (R₁=Br,R₄=C₆H₅R₇=H)

The intermediate 1-1 (R₁=Br) was prepared by using 5-bronco-1-indanoneas starting material according to the procedure described in Example 1.

The intermediate 1-2 (R₁=Br, R₄=C₆H₅) was prepared according to theprocedure described in Example 3 by using 1-1 (R₁=Br) andhydrocinnamaldehyde as starting materials.

The intermediate 1-3 (R₁=Br, R₄=C₆H₅) was prepared according to theprocedure described in Example 6 by using 1-2 (R₁=Br, R₄=C₆H₅) asstarting material.

The 2-benzoyl-7-bromo-4H,9H-naphtho[2,3-b]furan-4,9-dione 3-2 (R₁=Br,R₃=C₆H₅, R₇=H) was obtained according to the procedure described inExample 14 by using 1-3 (R=Br, R₄=C₆H₅) as starting materials with yieldof 25%, ¹H NMR (in CDCl₃) δ 7.58 (t, J=8, 2H). 7.67-7.72 (m, 2H),7.93-7.96 (m, 1H), 8.09-8.12 (m, 3H), 8.4 (d, 1H).

EXAMPLE 16 Preparation of2-Acetyl-7-chloro-4H,9H-naphtho[2,3-b]furan-4,9-dione 3-2 (R₁=Cl,R₃=CH₃, R₇=H)

The 2-acetyl-7-chloro-4H,9H-naphtho[2,3-b]furan-4,9-dione 3-2 (R₁=Cl,R₃=CH₃, R₇=H) was obtained according to the procedure described inexample 13 by using 2-ethyl-7-chloro-4H,9H-naphtho[2,3-b]furan-4,9-dione1-3 (R₁=Cl, R₄=CH₃) as starting materials with yield of 30%. ¹H NMR (inCDCl₃) δ 2.67 (s, 3H), 7.61 (s, 1H), 7.74-7.78 (m, 1H), 8.17-8.23 (m,2H).

EXAMPLE 17 Preparation of2-Acetyl-7-fluoro-4H,9H-naphtho[2,3-b]furan-4,9-dione 3-2 (R₁=F, R₃=CH₃,R₇=H)

The 2-acetyl-7-fluoro-4H,9H-naphtho[2,3-b]furan-4,9-dione 3-2 (R₁=F,R₃=CH₃, R₇=H) was obtained according to the procedure described inexample 13 by using 2-ethyl-7-fluoro-4H,9H-naphtho[2,3-b]furan-4,9-dione1-3 (R₁=F, R₄=CH₃) as starting materials with yield of 30%, ¹H NMR (inCDCl₃) δ 2.67 (s, 3H), 7.44-7.49 (m, 1H), 7.61 (s, 1H) 7.90-7.93 (m,1H), 8.25-8.30 (in, 1H).

EXAMPLE 18 Preparation of2-Acetyl-3-bromomethyl-4H,9H-naphtho[2,3-b]furan-4,9-dione 4-6 (R₁=H,R₃=—CH₃, and R₇=—CH₂Br)

To a solution of 5 grain (0.0594 moles) of 3-penten-2-one in 100 ml ofpentane in ice bath with vigorously stirring, was slowly added 9.5 grams(0.0594 moles) of bromine in 20 ml of pentane within 30 minutes. Afterstirred for additional 5 minutes in ice bath, the mixture was evaporatedto remove most of pentane. The small volume of 3,4-dibromo-2-pentanoneresidue from step 1 was dissolved in 200 ml of THF, and then chilled inan ice bath. To the solution in ice bath with vigorously stirring, wasslowly added 9.0 grams (0.0594 moles) of DBU within 30 minutes. Largequantity of precipitate salt was generated. The mixture was directlyused for next step reaction. To the reaction mixture of3-bromo-3-penten-2-one, 10.4 grams (0.0594 moles) of2-hydroxy-1,4-naphthoquinone was added. The resulting mixture wasstirred vigorously in a room temperature water bath. Then 9.9 grams(0.0650 moles) of DBU in was slowly added to the mixture within 30minutes. The temperature of the reaction mixture rose by the heatgenerated from reaction and was controlled to below 35° C. by adding iceto the water bath. After vigorously stirred for additional 3 hours underair at room temperature, the mixture was evaporated to small volume,then 500 ml of water was added to the residue. The resulting mixture wasextracted with dichloromethane. The organic phase was washed with water,aqueous 5% sodium bicarbonate and water, respectively, and then driedwith sodium sulfate. 200 mg of2-Acetyl-3-methyl-4H,9H-naphtho[2,3-b]dihydrofuran-4,9-dione wasobtained by silica gel purification.2-Acetyl-3-methyl-4H,9H-naphtho[2,3-b]dihydrofuran-4,9-dione 4-5 (R₁=H,R₃=R₇=CH₃) ¹H NMR (in CDCl₃) δ 1.55d, J=7, 3H), 2.35 (s, 3H), 3.58 (m,1H), 4.75 (d, J=7, 1H), 7.69-7.77 (m, 2H), 8.06-8.12 (m, 2H). Thepurified dihydrothran intermediate was dissolved in dichloromethane. Tothe solution, 300 mg of bromine was added and the resulting mixture wasstirred at room temperature overnight. The mixture was evaporated tosmall volume and loaded onto silica gel column. The desired pure2-acetyl-3-bromomethyl-4H,9H-naphtho[2,3-b]furan-4,9-dione 4-6 (R₁=H,R₃=CH₃, R₇=BrCH₂) was obtained. ¹H NMR (in CDCl₃) δ 2.78s, 3H), 4.51 (s,2H), 7.80-7.83 (m, 2H), 8.21-8.27 (m, 2H).

EXAMPLE 19

Biological Assays

Compounds of the present invention can be tested according to theprotocol described above. Table 2 shows the list of compounds describedin the protocol.

TABLE 2 Structure Compound

411

412

413

102

414

415

103

302

416

105

401

402

403

101

301

405

407

408

409

410

303

420

417

201

418

419

106

108

202

304

305

1001

404

Cell Culture: HeLa, DU145, H1299, DLD1, SW480, A549, MCF7, LN18, HCT116,HepG2, Paca2, Panel, LNcap, FaDu, HT29, and PC3 cells (ATCC, Manassas,Va.) were maintained in Dulbecco's Modified Eagle Medium (DMEM)(Invitrogen, Carlsbad, Calif.) supplemented with 10% fetal bovine serum(MS) (Gemini Bio-Products, West Sacramento, Calif.) and 5%penicillin/streptomycin/amphotercin B (Invitrogen).

Luciferase Reporter Assay: HeLa Cells were co-transfected withStat3-luciferase (Stat3-Luc) reporter vector (Panomics, Fremont, Calif.)and Renilla luciferase (Promega, Madison, Wis.) using Lipofectamine 2000as described by the manufacturer (Invitrogen). Following transfection,cells were maintained in medium containing 0.5% FBS for 24 hours. Cellswere then treated with the indicated compound for 30 minutes prior tothe addition of 25 ng/ml oncostatin M (OSM) (R&D Systems, Minneapolis,Minn.) to the medium. 6 hours following OSM addition, cells wereharvested and levels of firefly and renilla luciferase were measuredusing the Dual-Glo Luciferase Assay System as described by themanufacturer (Promega).

STAT3 DNA Binding Assay: Electrophoretic mobility shift assay (EMSA) wasperformed as described by the manufacturer (Li-Cor Biosciences, Lincoln,Nebr.). Briefly, nuclear extracts were made from HeLa cells using theNucBuster Protein Extraction Kit as described by the manufacturer (EMDBiosciences, San Diego, Calif.). 5 μg of nuclear extract waspre-incubated with the indicated dose of indicated compound for 30minutes prior to a 15-minute incubation with the IR700-labeled consensusStat3 oligonucleotide. Samples were then electrophoresed on apolyacrylamide gel and directly scanned using the Odyssey infraredimaging system (Li-Cor Biosciences). For the enzyme-linked immunosorbentassay (ELISA), 5 μg of nuclear extract was preincubated with indicatedconcentration of indicated compound for 30 minutes prior to the additionof biotinylated oligo (5′-Biotin-GATCCTTCTGGGAATTCCTAGATC-3′). Stat3-DNAcomplexes were then captured on streptavidin coated 96 well plates(Pierce, Rockford, Ill.). Bound complexes were then incubated with Stat3polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.)followed by anti-rabbit HRP conjugated secondary antibody (GEHealthcare, Pittsburgh, Pa.). Bound antibody was then visualized byaddition of TMB substrate (Pierce) and absorbance measured at 450 nm.

Cell Viability Determination: For 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) (Sigma-Aldrich, St.Louis, Mo.) analysis, cells were plated in 96 well plates at 10,000cells per well. 24 hours after plating, compound was added to cells atindicated doses. 22 hours following compound addition, MTT was added toeach well (0.5 mg/ml, final concentration) and plates were incubated foran additional 2 hours at 37° C., Medium was then aspirated and theformazan product was solubilized in 100 μl of isopropyl alcohol. Theabsorbance of each well was measured at 570 nm using a microplatereader.

Hoechst Side Population: To identify and isolate side population (SP)and non-SP fractions, SW480 cells were removed from the culture dishwith trypsin and EDTA, pelleted by centrifugation, washed withphosphate-buffered saline (PBS), and resuspended at 37° C. in Dulbecco'smodified Eagle's medium (DMEM) containing 2% FBS and 1 rnM HEPES. Thecells were then labeled with Hoechst 33342 (Invitrogen) at aconcentration of 5 μg/mL. The labeled cells were incubated for 120minutes at 37° C., either alone or with 50 μM verapamil (Sigma-Aldrich,St. Louis). After staining, the cells were suspended in Hanks' balancedsaline solution (MISS; Invitrogen) containing 2% FBS and 1 mM HEPES,passed a through 40 μm mesh filter, and maintained at 4° C. until flowcytometry analysis. The Hoechst dye was excited at 350 nm, and itsfluorescence was measured at two wavelengths using a 450 DF10 (450/20 nmband-pass filter) and a 675LP (675 nm long-pass edge filter) opticalfilter. The gating on forward and side scatter was not stringent, andonly debris was excluded [26].

CSC isolation with surface markers: Sorting tumor cells based primarilyupon the differential expression of the surface marker(s), such as CD44or CD133, have accounted for the majority of the highly tumorigenic CSCsdescribed to date. CD133 isolation is based upon the method ofRicci-Vitiani et al. [31], with slight modification. CD133⁺ cells wereisolated by either fluorescence activated cell sorting (FACS) ormagnetic nanoparticle-based separation. Briefly, 10⁷ cells/mL werelabeled with CD133/1 (AC133)-PE for FACS-based cell sorting; or withCD133/1 (AC133)-biotin (Miltenyi Biotec, Auburn, Calif.) for magneticfield-based separation using the EasySep® biotin selection kit (MiltenyiBiotec) according to the manufacturer's recommendations. Non-specificlabeling was blocked with the supplied FcR blocking reagent and antibodyincubations (1:11) were carried out on ice for 15 minutes in PBS with 2%FBS and 1 mM EDTA. Five washes were done for EasySep® isolation, whereascells were pelleted at 400 ×g for 5 minutes and resuspended at 2×10⁷/mL,before sorting by FACS.

CD44^(high) cells were isolated by FACS according to the methodsdescribed in Ponti et al., with slight modification [82]. Briefly, aftertrypsinization and recovery of cells for 30 minutes at 37° C. in growthmedia, cells were pelleted at 400×g and were resuspended in PBS with 2%FBS and 1 mM EDTA at 1×10⁶ cells/mL. Cells were then incubated on icewith a 1:100 dilution of CD44-FITC (BD Biosicences, San Diego, Calif.)for 15 minutes. Alternatively, CD24-PE (BD Bioscences, San Diego,Calif.) (1:100) was utilized for negative selection. After washing threetimes, cells were resuspended at 2×10⁶/mL and passed through a 40 μMmesh before sorting

Sphere assay: A reliable method of measuring the self-renewal capacityof cell population if the ability to be cultured as spheres in theabsence of serum or attachment. CD44^(h)t FaDu or Hoechst sidepopulation cancer stem cells were cultured in ultra low attachmentplates in cancer stem cell media (DMEM/F12, B27 Neurobasal supplement,20 ng/ml EGF, 10 ng/ml FGF, 4 μg/ml insulin, and 0.4% BSA) to allowspheres formation. Typically, sphere formation was evaluated bymicroscopy after 10-14 days in culture and spheres with >50 cells werescored.

EXAMPLE 20

Identification of Compounds that Selectively Kill a Broad. Spectrum ofCancer Cells

Identification of compounds that are apoptotic to a broad spectrum ofcancer cells in vitro. Cells plated in 96 well plates and treated withindicated compounds were subjected to MTT analysis at 24 hours followingcompound treatment to determine cell viability. IC₅₀ values calculatedacross multiple cell lines are summarized in Table 3 and Table 4 below.The data demonstrate that these compounds have potent activity againstbroad spectrum of cancer cells.

TABLE 3 IC₅₀ (μM) Cell Line Tissue #401 #402 #412 #416 #418 A549 Lung0.95 3.16 1.90 1.06 H1299 Lung 0.23 1.04 0.52 0.25 0.34 MCF7 Breast 0.461.15 0.75 0.46 HeLa Cervix 0.43 2.01 1.69 0.62 0.80 DLD1 Colon 0.33 2.511.11 0.54 0.64 SW480 Colon 0.32 1.49 1.31 0.44 0.76 HCT116 Colon 0.582.02 0.69 0.61 HT29 Colon 1.27 4.64 1.91 1.83 HepG2 Liver 0.25 Paca2Pancreas 0.11 0.49 0.64 0.21 0.21 Panc1 Pancreas 1.70 7.21 3.69 2.591.54 DU145 Prostate 0.12 0.55 0.33 0.22 0.18 PC3 Prostate 2.37 8.48 4.453.10 3.04 LNCap Prostate 0.63 FaDu Head and Neck 0.39

TABLE 4 IC₅₀ (μM) IC₅₀ (μM) IC₅₀ (μM) IC₅₀ (μM) Compound # DU145 H1299Hela FaDu 401 0.116 0.234 0.428 0.39 402 0.554 1.039 2.013 403 6.5 4 1013.7 3-10 11.7 301 0.835 0.794 3.358 405 1.405 407 2.105 4.113 3.779 4080.554 3.617 4.471 409 0.442 1.033 1.880 410 0.239 1.876 2.515 411 0.61614.052 14.748 412 0.327 0.524 1.689 413 0.721 1.897 4.375 102 11.418 41414.092 11.315 13.031 415 9.8 6.5 10.5 103 >10 8.9 16.7 302 4.9 7.2 7.8416 0.211 0.337 0.711 105 6.5 8.4 13 303 2.881 417 0.768 201 1.756 4180.164 0.317 0.488 419 1.822 106 4.84 108 30.848 20.713 202 2.645 4.558304 1.841 1.67 305 1.104 1.707 420 3.5

EXAMPLE 21

Identification of Stat3 as an Anti-Cancer Stem Cell Target

Stat3 knockdown in CSCs induces apoptosis. To determine whether cancerstem cells expressed Stat3 and whether Stat3 was constitutively active,we performed immunofluorence microscopy, which allows not only theanalysis of rare cell populations, but also provides additionalinformation on protein localization and the ability to correlatestaining with phenotype (i.e. apoptosis). Following immunofluorescentdetection of p-Stat3 and Stat3 in NSP and SP cells isolated by FACS fromSW480 colon cancer cells, we determined that Stat3 was indeed present inSP cells and that it was modestly enriched in the nucleus (FIG. 3A). Inaddition, we also observed increased p-Stat3 staining in SP cells overNSP cells, suggesting that SP cells may rely more heavily on Stat3 forsurvival.

The status of Stat3 was also evaluated in CD133⁺ cells isolated fromFaDu human head and neck cancer cells and LN18 human glioblastornacells. As shown in FIG. 3B, Stat3 are also constitutively active inthese cells. Taken together, these data suggest Stat3 as a target thatis particularly important for cancer stem cells.

We next tested the effect of Stat3 knockdown in CSCs using TPIV®.Immunofluorescence analysis revealed that significant depletion of Stat3could be achieved within 24 hours of infection (FIG. 4A) on freshlyislolated CSCs (SP) and found that the majority of cells treated withStat3 TPIV® underwent apoptosis within 24 hours of infection, whereascontrol TPIV® did not induce apoptosis to levels above control,uninfected cells (FIG. 4B). These data demonstrate that cancer stemcells depend upon Stat3 for survival.

Knock down Stat3 in CSCs inhibits CSC spherogenesis.CD44^(high)/CD24^(low) FaDu or Hoeschst side population cancer stemcells were isolated by FACS, and cultured in ultra low attachment platesin cancer stem cell media (DMEM/F12, B27 Neurobasal supplement, 20 ng/mLEGF, 10 ng/mL FGF, 4 μg/mL insulin, and 0.4% BSA) to allow sphereformation. Primary spheres were collected, disaggregated with trypsin,and distributed to 96-well ultra low attachment plated prior to TPIV®treatment. Bacteria were administered at an MOI of 1000 for two hoursbefore addition of antibiotic cocktail (penstrep, gentamycin,oflaxacin). Sphere formation was assessed after 10-14 days in culture.Representative sphere images were captured before (FIG. 5, left upperpanels) or after the addition of trypan blue to identify dead cells(FIG. 5, left bottom panel). Relative spherogenesis was shown in theright panel of FIG. 5. The data clearly showed that Stat3 knockdown incancer stem cells inhibits sphereogenesis, demonstrating that Stat3 is akey self-renewal factor of cancer stem cells.

EXAMPLE 22

Identification of Compounds that Inhibit Stat3 Pathway Activity

Inhibition of Stat3 transcription activity. Compounds were tested fortheir ability to inhibit Stat3 transcription activation activity incells using a Stat3-luciferase (Stat3-luc) reporter construct. Cellstransfected with Stat3-luc were cultured in reduced serum medium priorto addition of indicated compound for 30 minutes. Cells were thenstimulated with 25 ng/ml oncostatin (OSM) for 6 hours followed bydetection of Stat3-luc reporter activity. Compounds tested in the Stat3luciferase reporter assays and the results are summarized in Table 5.

TABLE 5 Compound # IC₅₀ in Stat3-Luc assays 401 ~0.25 μM 416 ~0.75 μM418 ~0.75 μM 402 ~0.75 μM 412 ~0.75 μM 410 ~1 μM 409 ~2 μM 408 ~2 μM 301~2 μM 407 ~5 μM

Inhibition of Stat3 DNA-binding activity. Nuclear extracts from HeLacells, which contain constitutively activated Stat3 as detected byphoshporylation at the tyrosine 705 residue, were used to perform Stat3EMSAs to monitor Stat3 DNA binding activity. Nuclear extracts wereincubated with indicated compound prior to incubation with IR700-labeledStat3 consensus oligonucleotide. Binding of Stat3 to the oligonucleotidewas monitored by gel electrophoresis and detection using a LiCor Odysseyinfrared scanner. The Stat3 retarded band was identified and confirmedby supershift with the anti-Stat3 antibody (FIG. 6A, left panel) anddose-dependent inhibition with the Stat3 peptide (FIG. 6A, middlepanel). Dose dependent inhibition of Stat3 DNA binding was observedfollowing incubation of the labeled probe with compound 401 (FIG. 6A,right panel).

Additional compounds were tested in the IMSA assays and the results areshown in FIG. 6B and Table 6. To calculate the inhibition %, the densityof the untreated Stat3 retarded band (control) was set as 100 and theinhibition % was the difference between the control and the relative DNAbinding activity of the drug-treated samples. These data shows that thecompounds of this invention can inhibit Stat3's DNA binding activity.

TABLE 6 inhibition % Compound 1 × MTT IC₅₀ 3 × MTT IC₅₀ 418 39 63 420 4763 302 6 25 106 26 51 202 50 46 402 29 57 301 20 26 406 16 34 103 98 26304 25 55

EXAMPLE 23

Identification of Compounds that Target Cancer Stem Cells

In order to test compounds for anti-CSC activity, freshly isolated CSCs(SW480 Hoechst SP cells or CD44^(high) FaDu cells) were exposed to adose range (30-0.117 μM) of compound for 48 h before examining cellviability by MTT assay. IC₅₀s were estimated by plotting the percentageof surviving cells. As shown in Table 7 and Table 8, the compounds ofpresent invention can target cancer stem cells.

TABLE 7 IC₅₀ (μM) Compound NSP SP 420 0.51 0.59 401 0.33 0.34 418 0.330.34 402 0.38 0.4 302 1.29 2.06 106 2 4.44

TABLE 8 IC₅₀ (μM) Compound CD44^(low) CD44^(high) 202 2.25 2.4 304 2.492.41 302 3.68 0.68

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The invention claimed is:
 1. A compound selected from the groupconsisting of:

or a tautomer, or pharmaceutically acceptable salt thereof.
 2. Apharmaceutical composition comprising a compound of formula IV,

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptablesalt thereof, wherein the symbols have the following meanings and are,for each occurrence, independently selected: X is O or S; R₁ ishydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or substituted alkyl,alkenyl or substituted alkenyl, alkynyl or substituted alkynyl,cycloalkyl or substituted cycloalkyl, cycloalkenyl or substitutedcycloalkenyl, heterocycle or substituted heterocycle, aryl orsubstituted aryl, OR_(a), or SR_(a); R₇ is hydrogen, halogen, cyano,nitro, CF₃, OCF₃, alkyl or substituted alkyl, alkenyl or substitutedalkenyl, alkynyl or substituted alkynyl, cycloalkyl or substitutedcycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle orsubstituted heterocycle, aryl or substituted aryl, OR_(a), or SR_(a); R₈is hydrogen, substituted alkyl, cycloalkyl or substituted cycloalkyl,aryl or substituted aryl, alkylaryl or substituted alkylaryl; R₉ and R₁₀are each independently hydrogen, alkyl or substituted alkyl, cycloalkylor substituted cycloalkyl, alkylaryl or substituted alkylaryl,alkylheteroaryl or substituted alkylheteroaryl; or R₉ and R₁₀ togetherwith the carbon to which they are bonded optionally form cycloalkyl orsubstituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl,heterocycle or substituted heterocycle; R_(a), is hydrogen, alkyl orsubstituted alkyl, alkenyl or substituted alkenyl, alkynyl orsubstituted alkynyl, cycloalkyl or substituted cycloalkyl, cycloalkenylor substituted cycloalkenyl, heterocycle or substituted heterocycle, oraryl or substituted aryl; and n is 1-4; and apharmaceutically-acceptable excipient, carrier, or diluent.
 3. A methodof treating a cancer in a subject, the method comprising administeringthe subject a therapeutically effective amount of a compound of formulaIV,

or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptablesalt thereof, wherein the symbols have the following meanings and are,for each occurrence, independently selected: X is O or S; R₁ ishydrogen, halogen, cyano, nitro, CF₃, OCF₃, alkyl or substituted alkyl,alkenyl or substituted alkenyl, alkynyl or substituted alkynyl,cycloalkyl or substituted cycloalkyl, cycloalkenyl or substitutedcycloalkenyl, heterocycle or substituted heterocycle, aryl orsubstituted aryl, OR_(a), or SR_(a); R₇ is hydrogen, halogen, cyano,nitro, CF₃, OCF₃, alkyl or substituted alkyl, alkenyl or substitutedalkenyl, alkynyl or substituted alkynyl, cycloalkyl or substitutedcycloalkyl, cycloalkenyl or substituted cycloalkenyl, heterocycle orsubstituted heterocycle, aryl or substituted aryl, OR_(a), or SR_(a); R₈is hydrogen, substituted alkyl, cycloalkyl or substituted cycloalkyl,aryl or substituted aryl, alkylaryl or substituted alkylaryl; R₉ and R₁₀are each independently hydrogen, alkyl or substituted alkyl, cycloalkylor substituted cycloalkyl, alkylaryl or substituted alkylaryl,alkylheteroaryl or substituted alkylheteroaryl; or R₉ and R₁₀ togetherwith the carbon to which they are bonded optionally form cycloalkyl orsubstituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl,heterocycle or substituted heterocycle; R_(a), is hydrogen, alkyl orsubstituted alkyl, alkenyl or substituted alkenyl, alkynyl orsubstituted alkynyl, cycloalkyl or substituted cycloalkyl, cycloalkenylor substituted cycloalkenyl, heterocycle or substituted heterocycle, oraryl or substituted aryl; and n is 1-4; and wherein the cancer isselected from the group consisting of breast cancer, lung cancer,cervical cancer, colon cancer, prostate cancer, liver cancer, head andneck cancer, and pancreatic cancer.